Tachedjian et al.indd
Drug Target Insights 2007: 2 159–182 159
REVIEW
Correspondence: Gilda Tachedjian, Ph.D., Molecular Interactions Group, The Macfarlane Burnet Institute for
Medical Research and Public Health, GPO Box 2284, Melbourne, Victoria, 3001, Australia.
Tel: 61 3 9282 2256; Fax: 61 3 9282 2100; Email: gildat@burnet.edu.au
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm
Targeting Human Immunodefi ciency Virus Type 1 Assembly,
Maturation and Budding
Johanna Wapling1,2, Seema Srivastava1, Miranda Shehu-Xhilaga3,4
and Gilda Tachedjian1,2,3
1Molecular Interactions Group, Macfarlane Burnet Institute for Medical Research and Public Health,
Melbourne, Victoria, 3004, Australia. 2Department of Microbiology, Monash University, Clayton,
Victoria 3168, Australia. 3Department of Medicine, Monash University, Prahran, Victoria 3181,
Australia. 4Infectious Diseases Unit, Alfred Hospital, Prahran, Victoria 3181, Australia.
Abstract: The targets for licensed drugs used for the treatment of human immunodefi ciency virus type 1 (HIV-1) are confi ned
to the viral reverse transcriptase (RT), protease (PR), and the gp41 transmembrane protein (TM). While currently approved
drugs are effective in controlling HIV-1 infections, new drug targets and agents are needed due to the eventual emergence
of drug resistant strains and drug toxicity. Our increased understanding of the virus life-cycle and how the virus interacts
with the host cell has unveiled novel mechanisms for blocking HIV-1 replication. This review focuses on inhibitors that
target the late stages of virus replication including the synthesis and traffi cking of the viral polyproteins, viral assembly,
maturation and budding. Novel approaches to blocking the oligomerization of viral enzymes and the interactions between
viral proteins and host cell factors, including their feasibility as drug targets, are discussed.
Keywords: HIV-1, antiretroviral drugs, drug targets, assembly, maturation, budding, protease dimerization, reverse
transcriptase dimerization.
Introduction
HIV-1 is a major public health problem affecting an estimated 40 million individuals worldwide (www.
unaids.org). Although it has been over 20 years since HIV-1 was identifi ed as the etiologic cause of
acquired immune defi ciency syndrome (AIDS) an effective vaccine is not available. Thus, apart from
public health measures that aim at HIV-1 prevention, the only effective strategy for controlling HIV-1
infections and lowering HIV-1 transmission is the use of antiretroviral drugs either for the treatment or
prevention of infections.
Current antiretroviral drugs belong to four classes, the nucleoside/nucleotide reverse transcriptase
inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease (PR) inhibitors
(PI) and fusion inhibitors (Vivet-Boudou et al. 2006; De Clercq, 1998; Abdel-Rahman et al. 2002;
Manfredi and Sabbatani, 2006). NRTIs and NNRTIs are respectively, competitive and allosteric
inhibitors of the HIV-1 reverse transcriptase (RT) and act early in the viral life-cycle by blocking the
conversion of the viral RNA genome into a double stranded proviral DNA precursor (Shehu-Xhilaga
et al. 2005). Fuzeon (enfuvirtide or T20) is a peptide that also acts early in the virus life-cycle by
preventing viral entry through interaction with the gp41 transmembrane protein (Shehu-Xhilaga
et al. 2005). In contrast, PIs inhibit the late stage of virus replication by blocking the specifi c cleavage
of Gag and Gag-Pol polyproteins to mature structural proteins and enzymes (Shehu-Xhilaga et al.
2005). Early antiretroviral regimens consisted of one or two RTIs, which were delivered as sequen-
tial monotherapy and led to treatment failure (Piacenti, 2006). The advent of combination therapy,
or highly active antiretroviral therapy (HAART) since 1996 has been responsible for a dramatic
decrease in AIDS mortality (Palella et al. 1998). Current HAART regimens generally comprise three
antiretroviral drugs, usually two NRTIs and either a PI or an NNRTI (Yeni et al. 2002). While an
armoury of agents is available for the treatment of HIV-1 patients, new drugs and drug targets need
to be identifi ed due to drug toxicity and the eventual emergence of drug resistant strains to current
antiretroviral inhibitors (Clavel and Hance, 2004). Moreover, resistance to one drug normally results
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Drug Target Insights 2007: 2
in cross-resistance to inhibitors of the same class,
rendering a large number of agents to limited
clinical use (Clavel and Hance, 2004). Therefore,
the development or availability of new drugs such
as Fuzeon, the HIV-1 integrase inhibitor ralte-
gravir (MK-0518) (Grinsztejn et al. 2007) and the
CCR5 antagonist maraviroc (Stephenson, 2007)
that remain active against drug resistant virus is
essential for the continuing success of HAART
(Yeni, 2006).
The increased understanding of how HIV-1
reproduces and interacts with the host cell
machinery has resulted in the identifi cation of
potential drug targets, which can be exploited for
the development of new classes of inhibitors. Here
we describe strategies and agents that block the
late stages of HIV-1 replication including the
synthesis and traffi cking of viral polyproteins,
viral assembly, maturation and budding. Novel
approaches to blocking the oligomerization of
viral enzymes and the interactions between viral
proteins and host cell factors are discussed
including their feasibility as drug targets. While
peptidomimetic PIs act at the late stage of HIV-1
replication to block viral maturation, this
review will deal with agents that inhibit HIV-1
PR by novel mechanisms that are distinct to
these transition state mimetics that are competi-
tive inhibitors of the HIV-1 PR.
Late Stages of the HIV-1 Life Cycle
Following virus attachment, fusion and uncoating
the single stranded positive sense RNA genome
of HIV-1 is reverse transcribed by the viral RT
into a proviral DNA precursor in a reverse tran-
scription complex (RTC) containing viral and
possibly host cell factors (Fig. 1). The RTC
matures into a preintegration complex (PIC) and
traffi cs to the nucleus where the viral cDNA is
inserted into the host cell chromosome by the
HIV-1 integrase (IN) (Telesnitsky A. and Goff,
1997). The processes from entry up to and
including integration are defi ned as the early
steps in the viral life cycle. The late stage of
virus replication begins with transcription of the
viral mRNAs from the integrated provirus
(Fig.1). Singly and multiply spliced mRNAs
encode the HIV-1 envelope proteins and regula-
tory/accessory proteins, respectively (Rabson
and Graves, 1997). Pr55gag (Gag) and Pr160gag-pol
(Gag-Pol) polyproteins are translated from
unspliced mRNAs (Swanstrom, 1997). Formation
of two types of polyproteins from the same
unspliced mRNA is mediated by a ribosomal
frameshifting mechanism that brings the pol
sequence in the same reading frame as gag.
Perturbation of ribosomal frameshifting leads to
changes in the Gag and Gag-Pol ratio that is
detrimental to virus assembly, morphogenesis
and release (Swanstrom, 1997).
Gag encodes the viral structural proteins
matrix (MA), capsid (CA), nucleocapsid (NC),
p6 and two spacer peptides, p1 and p2. Gag-Pol
also encodes MA, CA and NC in addition to the
three viral enzymes, PR, RT and IN. After trans-
lation, Gag and Gag-Pol are targeted to the host
cell plasma membrane, a process that is depen-
dent on the myristoylation of the N-terminus of
Gag (Fig. 1) (Swanstrom, 1997). Inhibition
of myristoylation disrupts the proper targeting of
Gag and Gag-Pol to the plasma membrane
(Swanstrom, 1997).
Gag-Gag, Gag/Gag-Pol and Gag-RNA interac-
tions are also essential for the proper assembly
and maturation of infectious virions. Gag and
Gag-Pol assemble at the plasma membrane along
with viral envelope glycoproteins gp120 and gp41
to form immature viral particles (Fig. 1). Gag is
necessary and suffi cient for virus particle forma-
tion (Freed, 1998; Swanstrom, 1997). The viral
genomic RNA is also packaged into virions
through interactions with the NC of Gag and a psi
packaging signal in the genome (Swanstrom,
1997). As the newly assembled virions bud from
the cell it is believed that Gag-Pol polyproteins
oligomerize in order to activate the HIV-1 PR by
forming an active PR homodimer. This results in
the sequential cleavage of Gag and Gag-Pol into
the mature structural proteins and enzymes
(Kaplan et al. 1994; Pettit et al. 1998). Agents that
bind to domains in Gag or Gag-Pol and modulate
their oligomerization are likely to have a negative
effect on virus assembly, maturation and budding
(Fig. 1). Agents that interfere with HIV-1 PR
mediated cleavage of Gag and Gag-Pol result in
the production of immature viral particles that are
non-infectious (Kohl et al. 1988).
Virus particle budding and egress is mediated
by interactions of viral proteins such as the p6 late
domain with components of the endosomal sorting
machinery. Ion channels formed by viral protein
U (Vpu) also facilitate viral particle egress from
the host cell. Below we describe in more detail the
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Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
specifi c processes required for viral assembly,
maturation and budding and agents that have been
described that block these steps.
Inhibitors of Gag and Gag-Pol
Expression: Targeting Ribosomal
Frameshifting
HIV-1 Gag and Gag-Pol polyproteins are encoded
by overlapping open reading frames on the same
unspliced mRNA. During translation Gag-Pol is
synthesized by a -1 ribosomal frameshifting
mechanism that occurs at a frequency of 5 to 10%
of Gag translation events (Jacks et al. 1988b).
Similar frameshifting mechanisms are also
used by other retroviruses including Rous
sarcoma virus and Mouse mammary tumor
virus in order to regulate expression of Gag-Pol
(Jacks and Varmus, 1985; Jacks et al. 1987;
Jacks et al. 1988a). The HIV-1 frameshift site is
a heptanucleotide AU-rich sequence (UUUUUUA)
found at the 3′ end of the NC coding sequence
and is conserved amongst HIV-1 isolates. This
slippery sequence and a downstream RNA stem
loop structure stall the ribosome during the
synthesis of Gag, allowing the ribosome to slip
Figure 1. Overview of the HIV-1 life-cycle. Early events in virus replication include attachment, fusion and uncoating of the virus followed
by reverse transcription in the cytoplasm of the cell, nuclear import of the preintegration complex and integration of the proviral DNA precur-
sor into the host cell chromosome. Late events begin with transcription of unspliced and spliced RNA from the provirus and export of the
mRNAs to the cytoplasm, resulting in the translation of Gag, Gag-Pol, Env and the accessory and regulatory proteins of HIV-1. Regulation
of Gag-Pol synthesis is mediated by a ribosomal frameshifting mechanism from unspliced mRNA that also expresses Gag. Myristoylation
of Gag is necessary for traffi cking of Gag and Gag-Pol to the site of viral assembly. Assembly is driven by interactions between Gag-Gag,
Gag/Gag-Pol, Gag-RNA. Viral budding and egress involves host cell factors. During or shortly after budding the HIV-1 PR cleaves the Gag
and Gag-Pol polyproteins resulting in a mature and infectious viral particle.
Late Event Targets
Frameshifting Myristoylation
& Trafficking
Assembly Budding & Egress Maturation
Early Events
Late Events
Recognition
& Attachment
Fusion
Uncoating &
Reverse Transcription
Nuclear
Import
mRNA
export
Translation
Myristoylation
& Trafficking
Maturation
Integration
Transcription
& Splicing
Assembly
Budding
& Egress
RTCPIC
Gag/Gag-Pol ratio Myristoylation Gag/Gag interactions
Gag/Gag-Pol interactions
Gag/RNA interactions
VPU
p6 PTAT motif
PR dimerization
RT dimerization
IN dimerization
Gag cleavage
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Wapling et al
Drug Target Insights 2007: 2
back one nucleotide and enable synthesis of the
Gag-Pol fusion protein (Jacks et al. 1988b). This
sequence, the stem-loop structure and its stability
and adjacent interacting sequences are believed
to be the key components of the frameshifting
signal (Jacks et al. 1988b; Kollmus et al. 1994;
Hill et al. 2005). Details of a recently reported
NMR structure and an analysis of current HIV-1
frameshifting models have recently been reviewed
(Brierley and Dos Ramos, 2006).
Studies demonstrate that perturbation of the
Gag/Gag-Pol ratio result in major defects in virus
replication, suggesting that interfering with ribo-
somal frameshifting represents a viable drug
target. Alteration of the Gag/Gag-Pol ratio, by
engineering vectors with gag and pol genes in the
same open reading frame, results in major defects
in assembly and budding (Karacostas et al.
1993; Park and Morrow, 1991). The block in virus
assembly is partially overcome by inhibition
of the HIV-1 PR, suggesting that increased HIV-1
PR activity is responsible for the defect (Karacostas
et al. 1993). A later study, in which the impact of
decreasing the ratio of Gag/Gag-Pol on virion
production was determined by co-transfection of
plasmids expressing Gag and Gag-Pol alone
demonstrate that the maintenance of this ratio is
not only important for HIV-1 replication but also
for virion RNA dimer formation and stability
(Shehu-Xhilaga et al. 2001a). Furthermore, a
decrease in Gag-Pol translation results in major
defects in virus maturation and HIV-1 infectivity
(Dulude et al. 2006). The small molecule, 1,4-
bis-[N-(3-N,N-dimethylpropyl)amidino]benzene
tetrahydrochloride (RG501, Table 1), is thought
to enhance ribosomal frameshifting of HIV-1 by
binding to the RNA stem loop structure of the
ribosomal frameshifting signal resulting in
increased ribosomal pausing (Hung et al. 1998).
The imbalance in the resulting Gag/Gag-Pol
ratio is associated with inhibition of acute and
chronic HIV-1 infection in CCRF-CEM cells and
peripheral blood mononuclear cells.
Targeting Gag and Gag-Pol
Traffi cking
During the late phase of the viral life cycle, Gag
polyproteins are targeted to the plasma membrane,
where they are believed to colocalise to lipid raft
microdomains for assembly into immature virions
(Morikawa et al. 1996; Bryant and Ratner, 1990;
Bouamr et al. 2003; Ding et al. 2003; Holm et al.
2003; Tang et al. 2004). Membrane targeting of
Gag is mediated by the N-terminal myristoyl group
in concert with conserved basic amino acids at the
N-terminus of the MA domain of Gag (Bryant and
Ratner, 1990; Facke et al. 1993; Ono and Freed,
1999; Ono et al. 2000). Myristic acid is a saturated
14-carbon fatty acid, post transationally attached
to the N-terminal glycine of both Gag and Gag-Pol
(Veronese et al. 1988). Myristoylation of Gag but
not Gag-Pol is critical for targeting these polypro-
teins to the plasma membrane (Park and Morrow,
1992; Smith et al. 1993). Mutations that interfere
with Gag myristoylation inhibit viral budding and
misdirect virion assembly to the cytosolic fraction
(Gottlinger et al. 1989; Bryant and Ratner, 1990).
However, complete inhibition of Gag myris-
toylation is necessary to block HIV-1 budding
(Morikawa et al. 1996).
Myristoylation is a two-step process involving
activation of myristate to myristoyl-CoA by acyl-
CoA synthetase and transfer of the myristoyl
moiety from the myristoyl-CoA substrate to the
N-terminal glycine of Gag by the enzyme
N-myristoyltransferase (NMT) (Morikawa et al.
1996; Veronese et al. 1988). This pathway has been
utilized to deliver alternate myristoylation
substrates that perturb viral assembly.
The myristic acid analogue 12-azidododeca-
noic acid is a potent inhibitor of HIV-1 produc-
tion in acute and chronically infected T-cell lines,
exhibiting a maximum inhibitory effect between
10–50 µM at noncytotoxic concentrations,
however the mechanism of action is not defi ned
(Devadas et al. 1992). Another analogue,
4-oxatetra-decanoic acid, reduces HIV-1 replication
in a T-cell line at 18 µM (Langner et al.
1 9 9 2 ) . Heteroatom-substituted analogs of
myristic acid such as 12-methoxydodecanoate
(13-oxamyristate or 13-OxaMyr), 5-octyloxypen-
tanoate (6-oxamyristate or 6-OxaMyr), 11-ethyl-
thioundecanoic acid and 12-thioethyldodecanoic
acid act as alternate substrates for Gag myris-
toylation (Bryant et al. 1989; Bryant et al. 1991;
Parang et al. 1997) and can prevent membrane
binding of the modifi ed Gag proteins (Bryant et al.
1989; Bryant et al. 1991). Of the heteroatom substi-
tuted analogs, 13-OxaMyr is the most potent
inhibitor. 13-OxaMyr is added to Gag with an
effi cacy similar to that of myristate and alters
viral polyprotein processing, which is suggested
to be a consequence of inhibiting Gag and
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Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
Table 1. Inhibitors of the late stages of HIV-1 replication.
Inhibitor Description Reference
Ribosomal frameshifting
1,4-bis-[N-(3-N,N Small molecule (Hung et al. 1998).
dimethylpropyl)amidino]benzene
tetrahydrochloride
(RG501)
Myristoylation and Traffi cking
12-azidododecanoic acid Myristic acid analogue (Devadas et al. 1992)
4-oxatetra-decanoic acid (Langner et al. 1992)
12-methoxydodecanoate Heteroatom-substituted (Bryant et al. 1989)
5-ocytl-oxypentanoate myristic acid analogues (Bryant et al. 1991)
11-ethylthioundecanoic acid (Parang et al. 1997)
12-thioethyldodecanoic acid
5-cis-tetradecenoic acid Unsaturated 14-Carbon (Lindwasser and Resh, 2002).
(physeteric acid) fatty acids
5-cis,8-cis-tetradecenoic acid
(goshuyic acid)
Assembly—Gag/Gag Interactions
CAP-1 Small molecule (Tang et al. 2003)
PAATLEEMMTA CA derived peptide (Niedrig et al. 1994).
GPG-NH2 CA derived tripeptide amide (Hoglund et al. 2002)
CAI Peptide (Sticht et al. 2005)
(Ternois et al. 2005)
Maturation—Gag processing
3-0-(3′-3′-dimethylsuccinyl)-betulinic Small molecule (Li et al. 2003)
acid (Zhou et al. 2004)
(PA-457/bevirimat) (Sakalian et al. 2006)
electrophilic disulfi de-substituted NC Zn fi nger inhibitor (Rice et al. 1995)
benzamides (DIBAs) (Turpin et al. 1996)
1,2-dithiane-4,5-diol,1,1-dioxide NC Zn fi nger inhibitor (Rice et al. 1997)
(NSC 624151)
S-acyl 2-mercaptobenzamide thioester NC Zn fi nger inhibitor (Schito et al. 2006).
(SAMT)
Maturation—PR dimerisation
Ac-TLNF-OH PR C-terminal tetrapeptide (Zhang et al. 1991)
Pal-YDL-OH Modifi ed PR C-terminal (Schramm et al. 1999)
Pal-YD-(biphenylalaine)-OH lipopeptides
Pal-YDT-OH
Apam(2)-YD-thyroxine-OH (Dumond et al. 2003)
PQITL(GGG)CTLNF Glycine linked PR interface (Babe et al. 1992)
tetra-peptides
HO-FNLTS-NH-(CH2)n-N-PQITLW-OH Alkyl linked PR interface (Zutshi et al. 1997)
peptides (Ulysse and Chmielewski, 1998)
(Zutshi and Chmielewski, 2000)
(Continued)
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Wapling et al
Drug Target Insights 2007: 2
Molecular tongs Scaffold constrained PR (Bouras et al. 1999)
interface peptides (Breccia et al. 2003)
(Merabet et al. 2004)
(Hwang and Chmielewski, 2005)
(Bannwarth et al. 2006)
β-sheet peptide/peptidomimetic PR interface derived (Song et al. 2001)
peptidomimetics
PQITL-RKKRRQRRRPPQV-SFNF- PR C-terminal fusion (Davis et al. 2006)
C/ATLN (P27/P27A) peptide
BocFψ[CH2NH]FEF-NH-CH2-CO- Linked PR C-terminal (Uhlikova et al. 1996)
TLNF-OH tetrapeptide—active site (Skalova et al. 2003)
inhibitor
Pentaester 13e Didemnaketal A analogue (Fan et al. 1998)
Ursolic Acid Triterpene (Quere et al. 1996)
NHGRNLLTQI (S8) PR LES peptide (Broglia et al. 2005)
(Broglia et al. 2006)
IVQVDAEG (p51) Random peptide (Park and Raines, 2000).
Vpr-(spacer)-TLNF-OH Vpr, PR C-terminal fusion peptide (Cartas et al. 2001).
Maturation—RT dimerisation
[2′,5′-bis-O-(tert-butyldimethylsilyl)-β- Small molecule (Sluis-Cremer et al. 2000)
D-ribofuranosyl]-3′-spiro-5″-(4″-amino- (Rodriguez-Barrios et al.
1′,2″-oxathiole-2″,2″-dioxide) 2001)
thymine (TSAO-T)
N-(4-tert-Butylbenzoyl)-2-hydroxy-1- Small molecule (Arion et al. 2002)
naphthaldehyde hydrazone) (BBNH) (Sluis-Cremer and
Tachedjian, 2002)
(Himmel et al.)
KETWETWWTE (Pep-7) RT connection subdomain peptide (Morris et al. 1999)
(Depollier et al. 2005)
TLMALELKGKLLLAGLAPSAFLPLSFP Designed peptide targeting (Campbell et al. 2002)
EGL (TLMA2993) RT connection subdomain (Hosokawa et al. 2004)
Maturation—IN Dimerisation
INI 1 Host cell factor (Yung et al. 2001)
(Sorin et al. 2006)
(Ariumi et al. 2006)
(Kalpana et al. 1994)
Budding and Egress
5-(N,N-hexamethylene)amiloride (HMA) Amiloride analogue (Ewart et al. 2002)
5-(N,N-dimethyl)amiloride (DMA) (Ewart et al. 2004)
Vpu Binding protein (UBP) Host cell factor (Callahan et al. 1998)
(Handley et al. 2001)
(Harila et al. 2006)
(Neil et al. 2006)
TSG101 Host cell factor (Garrus et al. 2001)
(Demirov et al. 2002)
(Goila-Gaur et al. 2003)
zLLL/MG-132 Lactocystin Proteosome inhibitor (Schubert et al. 2000)
(Ott et al. 2003)
Abbreviations: Ac: acetylation; Pam: palmitoyl; Apam: 2-aminopalmitic acid
Table 1. (Continued)
Inhibitor Description Reference
165
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
Gag-Pol traffi cking (Bryant et al. 1991). In an
acutely infected T-cell line 13-OxaMyr reduces
HIV-1 replication in the 40–80 µM range (Bryant
et al. 1989). 13-OxaMyr also inhibits viral
production in chronically infected H9/IIIB cells,
which is consistent for an inhibitor that targets
the late stage of HIV-1 replication (Bryant et al.
1991). 13-OxaMyr exhibits a synergistic anti-
HIV-1 effect with AZT suggesting its potential
for use in combination therapy (Bryant et al.
1991). The therapeutic effi cacy of 13-OxaMyr
can be further enhanced by conjugation with
glycerophospholipid L-∝-phosphatidylethanol-
amine (Pidgeon et al. 1993). The selectivity of
these heteroatom analogs for the target protein
is dependent on the position of the substituted
heteroatom, thus they can be exploited as a
therapeutic antiretroviral strategy. Nevertheless,
heteroatom-substituted myristic acid analogs are
still expected to adversely affect a substantial
range of cellular processes that depend on protein
N-myristoylation (Lindwasser and Resh, 2002).
An alternative strategy for targeting Gag myris-
toylation is the exogenous treatment of cells with
unsaturated 14-carbon fatty acids including 5-cis-
tetradecenoic acid (14:1n-9, physeteric acid) and
5-cis,8-cis-tetradecadienoic acid (14:2n-6, goshuyic
acid) (Lindwasser and Resh, 2002). As lipid rafts
have preference for saturated fatty acids, treatment
with unsaturated analogs interferes with membrane
targeting of Gag and consequentially viral assembly
and production (Lindwasser and Resh, 2002).
These inhibitors also interfere with certain Src-
kinase mediated cellular pathways, although they
appear to have no effect on cell proliferation
(Campbell and Vogt, 1995). It is suggested that
direct dietary intake of physeteric acid and goshuyic
acid could be a useful therapeutic strategy for the
treatment of HIV-1 infections. However, the effect
of long term intake of these unsaturated fatty acids
and their effect on N-myristoylated signaling
proteins such as Src, G-proteins, Arf and heteroge-
neously N-acylated retinal proteins needs to be
assessed (Lindwasser and Resh, 2002).
Recent studies also indicate a role for phospha-
tidylinositide 4,5-bisphosphate [PI(4,5)P2] in
regulating Gag localization (Ono et al. 2004).
In HIV-1, binding of PI(4,5)P2 to the MA
domain in Gag activates the “myristyol switch”
and also acts as the point of membrane attachment
(Saad et al. 2006). The binding site of PI(4,5)P2
on MA is highly conserved amongst HIV-1 strains
and therefore represents an attractive antiviral
target (Shkriabai et al. 2006; Saad et al. 2006).
Targeting HIV-1 CA
CA plays an important role in the HIV-1 life-cycle
by promoting Gag-Gag interactions during virion
maturation. The N- and C-terminal domains of this
protein serve distinct functions. As shown by
mutational analysis, the N terminal domain of CA
(N-CA), otherwise known as the NTD, is respon-
sible for maintaining the proper conformation of
CA during the assembly process (Worthylake et al.
1999; Li et al. 2000). The C-terminal domain of
CA (C-CA) or the CTD, is critical for Gag-Gag
interactions during assembly and maturation
(Gamble et al. 1996; Gamble et al. 1997) and
described mutations in this region have major
consequences on virion maturation and infectivity
(von Schwedler et al. 2003; Ganser-Pornillos et al.
2004). The NMR structure of CA has demonstrated
that the protein consists mainly of seven α-helices,
two β-hairpins and a loop structure (Momany et al.
1996; Gitti et al. 1996). Five of the α-helices form
a coiled-coiled structure while one of the
β-hairpins is located on the surface of the N-terminal
domain of the protein (Momany et al. 1996). The
second β-hairpin is predicted to be formed after
cleavage by the HIV-1 PR (Tang et al. 2002).
Cleavage of CA from its neighbouring proteins is
necessary for core condensation and conical capsid
shell formation (Vogt, 1996; Wiegers et al. 1998).
Compounds that bind to these regions would be
expected to disrupt proper CA shell formation and
virion infectivity making CA an important and
attractive target for the development of antiretro-
viral agents.
A proof of concept study, demonstrating the
potential of inhibiting CA-CA interactions as an
antiretroviral target has been published (Tang et al.
2003). Computational high throughput screening
of a small molecule library and NMR analysis for
binding specifi city resulted in the identifi cation of
CAP-1 and CAP-2 which bind to an apical site on
the NTD of both immature and mature CA (Tang
et al. 2003). While CAP-2, is toxic to U1 cells,
CAP-1 reduces viral infectivity by 95% at
100 µM. The released virions lack cone shaped
cores and resemble viral particles that have been
observed in HIV-1 expressing mutations that
disrupt CA-CA interactions (Dorfman et al. 1994;
Reicin et al. 1996; von Schwedler et al. 2003;
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Wapling et al
Drug Target Insights 2007: 2
Lanman et al. 2003). Despite aberrant viral
morphology CAP-1 does not affect viral particle
release or proteolytic processing (Tang et al.
2003). CAP-1 and CAP-2 bind to a common site
within the NTD thus preventing CA-CA interac-
tions and proper Gag assembly.
Peptides derived from HIV-1 CA have also
been described to affect viral morphogenesis by
interfering with capsid formation (Niedrig et al.
1994). The synthetic peptide, PAATLEEMMTA,
inhibits HIV-1 replication in cell culture assays
at 20–200 µg/ml and results in the production
of immature and aberrant viral particles (Niedrig
et al. 1994). Tripeptide amides derived from
the carboxyl terminus of CA inhibit HIV-1
replication, with the three most potent peptides
interacting with CA as demonstrated by capillary
electrophoresis analysis (Hoglund et al. 2002).
Glycyl-prolyl-glycine-amide (GPG-NH2) inter-
feres with the formation of HIV-1 particles
with a normal conical core structure (Hoglund
et al. 2002). G-NH2 is an active metabolite of
GPG-NH2 indicating that the latter acts as a
pro-drug (Andersson et al. 2005). However, the
development of HIV-1 resistance to either G-NH2
or GPG-NH2 has been elusive suggesting that the
peptides mediate their effects through a host cell
or other factor (Andersson et al. 2004).
CAI, a small peptide selected by phage display
screening, acts as an inhibitor of the assembly of
immature Gag in vitro (Sticht et al. 2005; Ternois
et al. 2005). CAI binds to the C-terminus of CA
(Kd ~ 800 µM), thus preventing the necessary
conformational changes in CA that lead to the
formation of mature cores (Sticht et al. 2005).
The structure of CAI complexed with CA has
revealed that the CAI binding region is a highly
conserved hydrophobic pocket within the C
terminus of CA where the peptide forms an extra
α-helix, which binds to the four α-helices of CA
(Ternois et al. 2005). The resulting protein-
peptide complex is therefore a fi ve α-helix bundle
with reduced CA-CA dimerization contacts that
destabilizes the dimer interface. Binding of CAI
to the C-CA not only affects the assembly of the
immature capsid particles but also reduces
the amount of correctly assembled mature capsids
in vitro, thus acting as a promising two-step
inhibitor (Sticht et al. 2005).
The C-terminal domain of Gag in the context
of Gag-Pol is essential for its interaction with Gag
and its incorporation into the virion (Srinivasakumar
et al. 1995; Chiu et al. 2002; Chien et al. 2006).
This sequence includes a highly conserved “major
homology region” (MHR) in the CA domain of
Gag and the adjacent CA-SP1 (Srinivasakumar
et al. 1995; Chien et al. 2006). These sequences
are also critical for HIV-1 Gag assembly as they
drive Gag oligomerization. However, the magni-
tude of the virion incorporation defect of Gag-Pol
MHR deletion mutants varies between different
studies making the value of targeting this region
of Gag-Pol unclear with respect to inhibition of
the late stages of HIV-1 replication (Mammano
et al. 1994; Srinivasakumar et al. 1995; Chiu et al.
2002; Chien et al. 2006).
Sequences involved in Gag and Gag-Pol
interactions are assumed to be similar to those
involved in Gag-Gag interactions. However,
virions generated in the presence of CAP-1 are
unlikely to affect Gag/Gag-Pol interactions as
defects in proteolytic processing in the virus or
virion associated RT activity were not observed
(Tang et al. 2003). The proline rich region of p6
has also been implicated in the packaging of cleaved
Pol proteins into virions, which is suggested to be
mediated by host cell proteins (Dettenhofer and
Yu, 1999; Cen et al. 2004). Identifying the host
cell factor implicated in the virion incorporation
of cleaved Pol will be necessary for establishing
this process as a viable drug target.
Targeting HIV-1 NC
The HIV-1 NC (NCp7) contains two highly
conserved zinc fi nger motifs C-X2-C-X4-H-X4-C
(X, any amino acid). The zinc fi ngers of NC are
critical in the early and late stages of HIV-1
replication with mutations in the zinc chelating
amino acids resulting in formation of non-
infectious virus (Aldovini and Young, 1990).
The zinc fi ngers of NCp7 are required for initi-
ation, elongation and effi cient template switching
d u r i n g r e v e r s e t r a n s c r i p t i o n ( R o d r i g u e z -
Rodriguez et al. 1995; Tanchou et al. 1995).
NCp7 is also involved in HIV-1 genomic RNA
dimerization, IN cleavage activity and coats the
viral RNA genome protecting it from nucleases
(Lapadat-Tapolsky et al. 1993).
Given the critical role of NCp7 zinc fi ngers in
HIV-1 replication it is not surprising that agents
that covalently modify the zinc chelating residues
of NCp7 have been described as inhibitors of HIV-1
replication (Rice et al. 1995). The electrophilic
167
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
disulfi de-substituted benzamides (DIBAs) inactivate
cell free virus and inhibit the early and late stages
of HIV-1 replication by interfering with reverse
transcription and viral particle maturation (Rice
et al. 1995; Turpin et al. 1996). In the U1 cell
line treatment with DIBAs results in the inhibition
of virus particle release, processing of Gag, and
the production of virions with reduced infectivity
(Turpin et al. 1996). The defect in viral particle
release and maturation was attributed to the
formation of intermolecular cross-linkages
between the zinc fi ngers of adjacent Gag mole-
cules, thereby preventing effi cient cleavage by the
HIV-1 PR (Turpin et al. 1996).
T h e n o n - d i s s o c i a b l e t e t h e r e d d i t h i a n e
compound 1,2-dithiane-4,5-diol,1,1-dioxide,
(NSC 624151) also mediates similar defects in
Gag processing (Rice et al. 1997). Although
cellular proteins also contain zinc fingers, these
inhibitors appear to preferentially target retro-
viral zinc fingers. This may be explained by the
inaccessibility of these inhibitors to the appro-
priate cellular compartments where zinc finger
containing cellular proteins are located. The
in vivo anti-HIV-1 activity of zinc finger
inhibitors has been demonstrated in a transgenic
murine model where infectious HIV-1 is induced
from an integrated provirus (Schito et al. 2003).
A recent study in a nonhuman primate model
demonstrated a reduction in the levels of SIV/
DeltaB670 in peripheral blood mononuclear
cells during therapy with the zinc finger inhib-
itor, S-acyl 2-mercaptobenzamide thioester
(SAMT), although there was no effect on viral
load (Schito et al. 2006). Further studies are in
progress to optimise the bioavailability and
pharmacokinetics of this promising inhibitor.
Targeting HIV-1 PR
Much of our understanding of how the PR domain
in Gag-Pol is activated and the processing cascade
of Gag and Gag-Pol are due to the contributions
of Kaplan and colleagues (Kaplan et al. 1994; Pettit
et al. 2005). Strict regulation of PR function is
critical for effi cient production of mature viral
particles. Premature activation, partial inhibition,
or over-expression of HIV-1 PR leads to major
defects in viral assembly and the production of
non-infectious viral particles (Krausslich, 1991;
Kaplan et al. 1993; Karacostas et al. 1993). Hence
novel inhibitors designed to prevent or perturb PR
dimerization could potentially inhibit the mature
PR homodimer and the immature Gag-Pol
embedded PR.
Targeting the PR Dimer Interface
with Interface Peptides
HIV-1 PR is a homodimeric aspartyl protease
formed by the symmetrical association of two 99
amino acid subunits. The crystal structure reveals
a compact, predominantly β-strand structure with
a short α-helix region near the C terminus
(Wlodawer et al. 1989). Dimerization of the PR
monomers generates both the substrate-binding
pocket and the catalytic centre and is essential for
PR activity (Cheng et al. 1990). The PR dimer has
a dissociation constant of 50 nM and Gibbs free
energy of dimer stabilisation of 10 kcal/mol (25 oC,
pH 3.4). Nearly 75% of the binding energy is
contributed by the four-stranded β-sheet formed
by the N- and C-termini (Todd et al. 1998).The
four-stranded β-sheet comprising the N- and
C-termini from each PR monomer represents an
attractive drug target for the following reasons: 1.
It is the major stabilising region of the active dimer,
2. The region is relatively free of known PR resis-
tance mutations, 3. The sequence is highly
conserved in most HIV-1 and HIV-2 isolates and
4. It provides a unique target minimising potential
toxicity issues for eukaryotic aspartyl proteases
(Gustchina and Weber, 1991).
A standard methodology for analysing potential
PR inhibitors that prevent PR dimerization (disso-
ciative inhibition) or target and bind to the PR
active site (competitive inhibition) has been
described (Zhang et al. 1991). An example of a
dissociative inhibitor is the C-terminal tetrapeptide,
Ac-T-L-N-F, which exhibits activity in the micro-
molar range (Ki 45 µM) (Zhang et al. 1991). Other
studies have also shown the capacity of N- and
C-terminal peptides, or ‘interface’ peptides, to bind
to PR monomers and thus prevent PR dimerization
and activity (Babe et al. 1991; Franciskovich et al.
1993; Schramm et al. 1991; Schramm et al. 1996).
The identifi cation of these lead peptides provides
proof of concept that targeting the PR β-sheet
region constitutes a viable strategy for the develop-
ment of novel inhibitors of HIV-1 PR.
The potency of C-terminal tetrapeptides are
increased by truncation to a core tripeptide, amino
acid modifi cation, and the addition of a linear
hydrophobic moiety such as palmitoyl to the amino
168
Wapling et al
Drug Target Insights 2007: 2
HIV-1 PR monmer
N-terminal
C-terminal
peptide
peptide
scaffold
‘molecular tong’
terminus of the peptide. The lipid moiety is thought
to increase the dissociative activity of the peptides
by directing it to the hydrophobic PR interface
(Schramm et al. 1999). However, despite their
capacity to inhibit PR activity at low nanomolar
concentrations, these lipopeptides are poorly
soluble and susceptible to protein degradation.
Further modifi cations have been made to the lipo-
peptides by making them less peptide-like
(Cafl isch et al. 2000) and by modifying the lipid
moiety to increase their solubility while retaining
potency (Dumond et al. 2003).
Cross-linking interfacial peptides represent
another strategy, with the aim to increase the
affi nity of the peptides by presenting them in a
conformation similar to a PR monomer. The fi rst
interface tetrapeptides tethered with a glycine
linker display greater potency (PF1, IC50= 40 µM)
compared to free tetrapeptides (IC50 ≥ 150 µM)
(Babe et al. 1992). This approach has evolved to
linking peptides with fl exible alkyl tethers (Zutshi
and Chmielewski, 2000) and semirigid alkyl based
tethers (Ulysse and Chmielewski, 1998), which
increase the distance between the peptides to
approximate that of the PR termini in the dimer
(~10 Å). The conformational freedom of these
linked peptides was addressed by the use of
pyridinediol and naphthalene based molecu-
larly constrained scaffolds (Bouras et al. 1999;
Song et al. 2001; Merabet et al. 2004; Bannwarth
et al. 2006). Known as ‘molecular tongs’, these
compounds are designed to position the interface
peptides to clamp the termini of a PR monomer
(Fig. 2). These studies have culminated in a set of
optimised tongs with symmetrical peptidomimetic
sequences based on an optimised PR C-terminal
sequence. The tongs inhibit the activity of HIV-1
PR that are either sensitive or resistant to PIs with
Ki values from 0.4–4.8 µM in cell free assays
(Bannwarth et al. 2006).
Other variations on the theme of interface
peptides include combining the advantages of
lipopeptides and molecular tongs. Interface
peptides have been linked to lipophilic groups by
a rigid bicyclic guanidinium scaffold (Breccia et al.
2003). The most potent compound demonstrates
PR inhibitory activity similar to tethered peptides
and molecular tongs. Cross-linked interfacial
peptides have been designed to irreversibly inhibit
HIV-1 PR by formation of a disulfi de bond between
the peptide and the conserved PR residues C-95
and C-67, and demonstrate a Ki in the low micro-
molar range (Zutshi and Chmielewski, 2000). The
C-terminal tetrapeptide has also been tethered to
a peptidic PR active site inhibitor, combining both
dissociative and competitive methods of inhibition
in one molecule (Uhlikova et al. 1996).
Random peptides that are dissociative inhibitors
of HIV-1 PR have been described. The bacterio-
phage lambda repressor protein was utilised to
develop a powerful two-hybrid PR dimerization
assay. From a library of 5 × 108 random peptides,
300 were identifi ed as potential PR dimerization
inhibitors. The most potent peptide identifi ed, p52,
was a pure dissociative inhibitor with low Ki
of
780 nM (Park and Raines, 2000).
Ultimately, one of the major hurdles in devel-
oping peptidic inhibitors is to obtain a biologically
stable compound that can be delivered inside the
cell. One mechanism to achieve this is to fuse the
peptide to amino acid sequences that promote
either encapsidation into viral particles or entry
into the host cell. Virus protein R (Vpr) is a HIV-1
accessory protein packaged into virions by its
trans association with the Gag p6 motif. Both
viral and cellular proteins have been successfully
delivered into viral particles as Vpr fusion
proteins (Wu et al. 1997). Inhibition of HIV-1
replication has been reported by the fusion of Vpr
to viral PR recognition sequences (Serio et al.
2000). Expression of the PR C-terminal tetrapep-
tide as a Vpr fusion [Vpr-(spacer)-T-L-N-F-OH]
attenuates HIV-1 replication in chronically
infected cells and in single-round replication
assays (Cartas et al. 2001).
Most recently, inhibition of HIV-1 replication
has been demonstrated by delivering PR interface
peptides as a fusion peptide utilising the HIV-1 Tat
derived cell permeable protein transduction domain
Figure 2. A molecular tong bound to the C-terminus of the
HIV-1 PR monomer.
169
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
(Davis et al. 2006). Peptides P27/A are PR
dimerization inhibitors that inhibit the activity of
wild-type and drug resistant PR in cell free assays
with IC50 values in the 0.28–0.58 µM range. These
peptides are successfully delivered into chronically
HIV-1 infected cells and reduce viral particle
production. This was observed by a reduction in
p24, rather than inhibition of Gag processing which
suggests that the peptide may interact with the
Gag-Pol embedded PR and disrupt the ordered
processing of Gag-Pol leading a decrease in viral
particle production (Davis et al. 2006).
PR Folding Inhibitors
Local elementary structures (LES) are comprised
of strongly interacting, highly conserved amino
acids that are usually hydrophobic. These amino
acids are suggested to direct the folding of a protein
into its native conformation. Short peptides corre-
sponding to or mimicking the LES are hypothesised
to act as folding inhibitors, preventing the protein
achieving its native conformation (Broglia et al.
2005). A peptide has been identifi ed from a LES
in the HIV-1 PR (peptide S8, amino acids 83–93)
that inhibits PR activity with a Ki of 2.58 µM and
results in disorganisation of the PR secondary
structure by reducing the β-sheet content from 30%
to 14% (Broglia et al. 2005; Broglia et al. 2006).
Current efforts are directed towards developing a
shorter less hydrophobic peptide or mimetic based
on the S8 lead peptide.
Catalytically Inactive PR Subunits
as Dominant Negative Inhibitors
of PR activity
Catalytically inactive PR monomers act in a
dominant negative fashion to inhibit wild-type
HIV-1 PR by forming inactive heterodimers in
recombinant protein assays (Babe and Craik,
1991). When virus expressing a PR active site
mutation is co-transfected with wild-type HIV-1,
both viral replication and virus infectivity are
reduced (Babe et al. 1995). Computer modelling
has been used to successfully design an optimised
dominant-negative PR expressing D25K, G49W
and I50W (KWW) (McPhee et al. 1996), which
also reduces viral replication and infectivity
(Junker et al. 1996). Biochemical studies on recom-
binant dominant negative PRs confi rm that the
mechanism of action is by formation of inactive
heterodimers (Rozzelle et al. 2000). Interestingly,
the mutant PRs cannot homodimerize and they fold
only when expressed with wild-type PR. PR
heterodimers are also more stable that the wild-type
homodimer (Rozzelle et al. 2000). Hence inactive
PR heterodimers form the dominant species. Such
a dominant negative strategy for the inhibition of
HIV-1 PR would require in vivo delivery by a gene-
therapy system, the therapeutic use of which is
unlikely in the near future.
Non-Peptide Inhibitors of HIV-1 PR
A screen of a crude extract from the marine
organism magenta ascidian didemnum identifi ed
two didemnaketals, A (a bicyclic ketal) and B
(a linear heptaprenoid), that inhibit HIV-1 PR
activity with IC50 values of 2 µM and 10 µM,
respectively (Potts et al. 1991). These compounds
are unsuitable drug candidates, but have given rise
to a novel class of pentaesters, the most potent of
which is a dissociative inhibitor of PR with a Ki
of 2.1 µM (Fan et al. 1998).
A novel class of PR dimerization inhibitors were
identifi ed by searching the Cambridge structural
database for pharmacophores that mimic the action
of previously identified inhibitory interface
peptides (Quere et al. 1996). Several triterpene
structures were identifi ed, of which ursolic acid
acts as a dissociative inhibitor of PR with an IC50
of 2 µM. It has been suggested that triterpene could
provide another basic scaffold for building more
effective peptidomimetics. Interestingly, another
member of the triterpene family, PA-457, acts as a
novel inhibitor of HIV-1 maturation which is
discussed later in this review. A β-sheet mimetic
was tested for its ability to inhibit PR homodimer-
ization by perturbation of β-sheet formation (Song
et al. 2001). The β-sheet mimetic had a relatively
high IC50 of 30 µM and the method of inhibition
appears to be complex, however the structure
provides a non-peptidic lead compound for PR
inhibitors.
Targeting Gag Processing
The rate and the specifi city of Gag cleavage by the
HIV-1 PR is dependent on the amino acid
composition of the different cleavage sites
recognized by the viral PR (Swanstrom, 1997).
Based on the order of proteolysis by HIV-1 PR,
these sites are classified as primary (p2/NC),
secondary (MA/CA and p1/p6) or tertiary (CA/p2
170
Wapling et al
Drug Target Insights 2007: 2
or CA/SP2) cleavage sites. The lack of processing
of any of these sites by PR results in the formation
of aberrant particles (Swanstrom, 1997). In
particular, inhibition of cleavage at the CA/p2 site
has severe consequences for core formation,
stability and virion infectivity (Pettit et al.
1994; Wiegers et al. 1998; Pettit et al. 1998; Shehu-
Xhilaga et al. 2001b). The α-helical structure that
stretches between the C-terminus of CA and the
N-terminus of SP1 is critical for virion assembly
and p2 function (Accola et al. 1998). Clearly, these
PR cleavage sites are potential targets for
antiretroviral drug design.
In this regard, a compound that interferes with
viral maturation by blocking CA/p2 cleavage has
been identifi ed. 3-O-(3′,3′-Dimethysuccinyl)
betulinic acid (PA-457 or bevirimat) potently
inhibits HIV-1 maturation and infectivity
(Li et al. 2003; Zhou et al. 2004). PA-457
specifi cally blocks the cleavage of CA/p2 in cell
based (Li et al. 2003) and in cell free assays
(Zhou et al. 2005; Sakalian et al. 2006), thus
inhibiting core condensation and virion matura-
tion. Inhibition of Gag processing at the CA/p2
junction results in the generation of the uncleaved
p25 product in transfected cells at 0.1 µg/ml of
PA-457 (Li et al. 2003). Consistent with the
proposed mechanism, PA-457 resistant HIV-1
selected in long term cultures in the presence of
betulinic acid contain mutations in the regions
that fl ank the P-P’ scissile bond (Adamson et al.
2006; Zhou et al. 2006). These mutated sites in
Gag are recognized by the viral PR during
proteolysis. In addition, other single amino acid
substitutions have been identifi ed that confer
resistance to PA-457 and are exclusively located
either at the C terminus of CA or within the fi rst
three amino acids of the p2 spacer peptide
(Adamson et al. 2006). Interestingly, they all
conferred resistance independently and were
located within the boundaries of the CA/p2
proteins, a region well known to promote Gag
multimerization (Adamson et al. 2006). These
data suggest that there is more than one mechanism
by which these mutants have acquired resistance
to PA-457. PA-457 has successfully undergone
Phase 1 and 2 clinical trials and is currently in a
Phase 2b trial to test the effi cacy of different doses
of PA-457 in combination with approved HIV-1
inhibitors as part of an optimised regimen in
patients failing therapy due to the emergence of
drug resistant virus.
Targeting the RT Domain in Gag-Pol
Like other HIV-1 enzymes, RT subunits must
oligomerize to form an active enzyme. The
biologically relevant form that is present in the
virion is an asymmetric heterodimer comprised
of the p66 (66 kDa) and the p51 (51 kDa) subunits
(Jacobo-Molina et al. 1993; Kohlstaedt et al.
1992). The RT heterodimer is extremely stable
and has an extensive protein surface area (4800 Å2)
that is buried upon subunit dimerization. Ther-
modynamic measurements of the association
between the p66 and p51 RT subunits have esti-
mated Gibbs free energy of dimer stabilization of
approximately 10–12 kcal/mol–1, corresponding
to a dissociation constant of approximately
3 × 10–7 M (Venezia et al. 2006). For an extensive
review on HIV-1 RT dimerization see Srivastava
et al. 2006.
Regions both upstream and downstream of the
PR region in Gag-Pol have been investigated for
effects on PR activation (Bukovsky and Gottlinger,
1996; Partin et al. 1991; Louis et al. 1999). Large
deletions within or C-terminal truncations of RT
in the context of Gag-Pol result in an increase in
virion associated Gag processing intermediates,
suggesting a defect in PR activity (Cherry et al.
1998; Liao and Wang, 2004; Quillent et al. 1996).
These studies suggest that modulating RT dimer-
ization in the context of Gag-Pol may have a
negative impact on PR activation and HIV-1
maturation.
The importance of the RT region in Gag-Pol
for both RT maturation and viral particle produc-
tion has been demonstrated by the study of RT
point mutations that prevent RT heterodimeriza-
tion and p66 homodimerization. Mutations at
W401, a component of the highly conserved
tryptophan repeat motif in the connection subdo-
main, blocks RT dimerization in vitro (Tachedjian
et al. 2003; Tachedjian et al. 2005b). When
expressed in HIV-1 it manifests as defects in
reverse transcription, aberrant processing of RT,
and low levels of infectivity (Wapling et al. 2005).
The L234A mutation, located in the RT primer
grip region, prevents RT dimerization and
decreases Gag-Pol stability (Tachedjian et al.
2000; Yu et al. 1998). This mutation reduces PR
incorporation into virions, increases the accumu-
lation of Gag processing intermediates, and
results in the production of non-infectious virus
particles (Tachedjian et al. 2000; Yu et al. 1998).
These examples demonstrate the potential for
171
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
targeting the Gag-Pol embedded RT for blocking
HIV-1 maturation.
Inhibitors that Modulate HIV-1 RT
Subunit Interaction
Apart from classical NNRTIs, there exists a class of
unconventional NNRTIs that bind to HIV-1 RT and
inhibit enzyme activity by decreasing the overall
stability of the heterodimer without dissociating the
complex (Sluis-Cremer et al. 2000; Sluis-Cremer and
Tachedjian, 2002; Sluis-Cremer et al. 2006; Camarasa
et al. 2006). The TSAO-T derivatives ([2′,5′-bis-O-
(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3′-
spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide)
thymine) destabilise both RT heterodimers and p66
homodimers by inducing changes at the RT dimer
interface (Sluis-Cremer et al. 2000; Rodriguez-
Barrios et al. 2001). The putative binding site is at
the RT dimer interface and overlaps in part with the
NNRTI binding pocket (Rodriguez-Barrios et al.
2001). Resistance mutations to the TSAO drugs are
readily generated in cell culture indicating that these
drugs are specific inhibitors of the HIV-1 RT
(Balzarini et al. 1993). While TSAO represent the
fi rst class of small molecules that destabilize the RT
heterodimer, preclinical studies demonstrate that the
pharmacological profile of TSAO inhibitors is
unfavourable for further clinical development
(Camarasa et al. 2006).
The N-acylhydrazone derivative N-(4-tert-
Butylbenzoyl)-2-hydroxy-1-naphthaldehyde
hydrazone (BBNH) binds to both the DNA poly-
merase and RNase H domains of RT and inhibits
both enzymatic activities of the RT (Arion et al.
2002). Similar to TSAO, BBNH prevents RT
activity through destabilizing, but not dissociating
the subunits. BBNH derivatives that bind to the
DNA polymerase domain alone are suffi cient to
induce dimer destabilization (Sluis-Cremer and
Tachedjian, 2002). The recently resolved structure
of HIV-1 RT bound to a BBNH derivative has
confi rmed that the binding site is in close proximity
to, but distinct from both the polymerase active
site and NNIBP. It is thought that BBNH destabi-
lizes the RT heterodimer by inducing changes in
the primer-grip motif, which is an important region
for RT dimer stability (Himmel et al. 2006;
Srivastava et al. 2006).
By targeting the RT dimerization interface, it is
possible that unconventional NNRTIs may have
an affect on the late stage of virus replication.
In particular, the TSAO drugs that destabilize the
p66 homodimer, may also perturb the process of
RT maturation to the heterodimer, and arguably
even target the RT domain in Gag-Pol and interfere
with PR activation. However, these possible effects
have not been described. Further elucidation of the
impact of nonclassical NNRTIs on RT maturation,
and the mechanism of destabilization would be
advantageous for designing more potent inhibitors
of both RT function and RT maturation.
Classical NNRTIs as RT Inhibitors
Acting at the Late Stage
of Viral Replication
Interestingly, several classical NNRTIs have been
shown to confer a concentration dependant
increase in RT heterodimer formation, corre-
sponding with a loss of RT polymerase function
(Tachedjian et al. 2001; Venezia et al. 2006). Efavi-
renz (EFV) is a strong enhancer of RT dimeriza-
tion, and also enhances the formation of p66 and
p51 homodimers (Tachedjian et al. 2005a). The
exact mechanism for increasing RT subunit inter-
actions is unknown but it is suspected that the
binding of EFV to the RT mediates conformational
changes in the p66 subunit that promotes interac-
tion with p51 (Tachedjian et al. 2001). EFV has
also demonstrated the capacity to enhance the
homodimerization of p66 in vitro, and a 90kDa
model Pol protein in an inducible bacterial expres-
sion system (Sluis-Cremer et al. 2004; Tachedjian
et al. 2005a). It has recently been demonstrated
that NNRTIs enhance Gag-Pol dimerization,
resulting in premature PR activation and a decrease
in viral particle release (Figueiredo et al. 2006). In
HIV-1 transfected cells, EFV, TMC120 and
TMC125 increased Gag and Gag-Pol processing,
and caused up to 45% decrease in viral particle
production. Similar effects were not observed for
NNRTIs that do not signifi cantly enhance p66
homodimerization and NRTIs (Figueiredo et al.
2006). Hence, NNRTIs that are potent enhancers
of RT dimerization also affect the late stage of viral
replication, which represents a novel inhibitory
mechanism for these drugs. However, the concen-
trations required to mediate this effect are two to
three orders of magnitude higher then concentra-
tions that block RT function. This is likely due to
reduced binding affi nity of the NNRTIs to the
proposed target which is the RT domain of Gag-Pol.
Strategies to identify drugs that are more potent
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Wapling et al
Drug Target Insights 2007: 2
inhibitors of this late stage in the viral life-cycle
could be identifi ed by screening for molecules that
enhance Gag-Pol dimerization. Such a screen could
be enhanced by incorporating mutations in the RT
that are known to confer decreased susceptibility
to current NNRTIs in order to select for drugs that
have the potential to block NNRTI resistant strains
of HIV-1.
Peptide Based Inhibitors
of RT dimerization
Two strategies have been utilised to generate peptides
designed to target the RT dimer interface in order to
block HIV-1 RT function, including peptides corre-
sponding to regions that are known to have an impor-
tant role in RT dimerization (Debyser and De Clercq,
1996; Depollier et al. 2005; Divita et al. 1994; Morris
et al. 1999b; Morris et al. 1999a). The most successful
of these peptides, Pep-7, corresponds to RT residues
395–404, derived from the highly conserved trypto-
phan repeat motif (W398–W414) (Depollier et al.
2005). Pep-7 interacts with p51, and destablizes both
the RT heterodimer and the p66 homodimer. Similar
to unconventional NNRTIs, Pep-7 is unable to induce
RT dissociation (Depollier et al. 2005). Pep-7 based
peptides are potent suppressors of HIV-1 replication
at noncytotoxic concentrations (Morris et al. 1999b).
The method of inhibition in HIV-1 infected cells has
not been elucidated. However, given that Pep-7 cannot
induce RT subunit dissociation, it has been suggested
that it acts at the late stage of virus replication by
preventing the formation of an active RT heterodimer
(Morris et al. 1999b).
Rational strategies utilising the available RT
structures to direct the design and manufacture of
mimetic peptides targeting subunit interaction is a
recent development (Campbell et al. 2002;
Hosokawa et al. 2004). These studies have led to
the synthesis of a peptide, TLMA2993, which also
targets the RT connection subdomain. TLMA2993
inhibits RT activity at micromolar concentrations
(Campbell et al. 2002). Cells stably transfected with
this peptide are protected from HIV-1 infection in
a concentration dependant manner due to inhibition
of reverse transcription, as observed by a decrease
in HIV-1 DNA (Hosokawa et al. 2004).
Targeting the IN Domain in Gag-Pol
IN catalyses the insertion of viral DNA into the
host chromosome and thus inhibits an early crucial
step in the virus life cycle. IN is also implicated in
reverse transcription, nuclear import of the
pre-integration complex, viral assembly and
budding (Engelman et al. 1995; Hehl et al. 2004).
Despite the numerous roles of IN in HIV-1 replica-
tion, new approaches for inhibiting viral replication
have focused on targeting the catalytic activity of
IN that is required for proviral DNA integration.
Two IN inhibitors, MK-0518 and GS-9137
(JTK-303) have entered clinical trials (Cotelle,
2006; Makhija, 2006) and have shown effi cacy in
Phase III clinical trials (Stephenson, 2007).
Since IN is expressed as part of Gag-Pol,
agents that bind to the IN domain in this polypro-
tein are likely to impact on the late stages of
replication. Consistent with this notion, mutations
in IN have been reported to effect virion formation
(Shin et al. 1994). Truncations of IN at the
C-terminus of Gag-Pol result in aberrant virion
core structures, with a reduction in the overall
levels of cell-associated viral Gag, suggesting a
defect in Gag-Pol processing (Engelman et al.
1995; Bukovsky and Gottlinger, 1996).
IN requires oligomerization for activity.
Therefore, inhibitors of IN function that mediate
their effects through negating IN subunit
interactions are also likely to interfere with viral
assembly. This would be manifested by interfering
with Gag-Pol/Gag-Pol interactions leading to
subsequent effects on HIV-1 PR activation
(Muriaux et al. 2004). In this regard peptide
inhibitors of IN dimerization have been reported,
however their effects on the late stages of the virus
life-cycle remains to be determined (Maroun et al.
2001; Zhao et al. 2003).
Certain host cell factors are incorporated into
the virion by interaction with the IN domain of
Gag-Pol. A cellular factor that has been implicated
in affecting the late stage of the virus life cycle is
integrase interactor 1 (INI1). INI1 was identifi ed
in a yeast two-hybrid screen for host cell proteins
interacting with HIV-1 IN (Yung et al. 2001;Yung
et al. 2004; Kalpana et al. 1994). INI1 mutants that
abrogate interaction with IN or cells defi cient in
INI1 exhibit a substantial reduction in viral produc-
tion (Yung et al. 2001). INI1 affects several steps
during HIV-1 replication (Ariumi et al. 2006; Sorin
et al. 2006; Yung et al. 2001) and is also packaged
into HIV-1 particles (Kalpana et al. 1994). A 110-
amino-acid fragment of INI1 (S6) with a minimal
IN-interaction domain inhibits viral production
(Yung et al. 2001; Yung et al. 2004). The inhibitory
effect of S6 on HIV-1 production is mediated by
173
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
binding of the ectopically expressed S6 to the
Gag-Pol embedded IN. Furthermore, stable
expression of a transdominant S6 mutant inhibits
infection in T-cells. S6 represents a potential lead
for the development of inhibitors of the late stage
of HIV-1 replication (Yung et al. 2001).
Proteosome Inhibitors
Intracellular degradation of misfolded, damaged
or unwanted proteins is mediated by the proteo-
some, which is a multisubunit proteolytic complex
of 26S (Schubert et al. 2000). Proteins are tagged
for proteolytic destruction by the covalent attach-
ment of a chain of ubiquitin polypeptides on lysine
residues of the protein (Schubert et al. 2000).
Proteosome inhibitors inhibit the late stages of the
HIV-1 life-cycle by interfering with viral particle
release and maturation (Schubert et al. 2000).
Decreased budding has also been demonstrated for
retroviruses expressing the PPPY- or PTAP
containing late domains but not those that use the
YPDL type late domain (Schubert et al. 2000; Ott
et al. 2003). The effect is not dependent on the viral
particle assembly site (i.e. cytoplasm or plasma
membrane) or on monoubiquitination of Gag (Ott
et al. 2003). In addition to a decrease in viral
particle release (4-fold), virions released from cells
treated with proteosome inhibitors have approxi-
mately a 10-fold decrease in infectivity (Schubert
et al. 2000). The impact of proteosome inhibitors
is dependent on an active HIV-1 PR and the pres-
ence of the p6 late domain but is independent of
Vpu function. Inhibition of HIV-1 maturation and
budding is observed with reversible (zLLL also
known as MG-132) and irreversible (lactocystin)
proteosome inhibitors (Schubert et al. 2000).
Proteosome inhibitors also interfere with the
activity of the HIV-1 viral infectivity factor (Vif) on
the antiviral function of apolipoprotein B mRNA-
editing enzyme catalytic polypeptide-like 3G
(APOBEC3G) in virus producing cells (Stopak et al.
2003; Sheehy et al. 2003; Mehle et al. 2004; Yu et al.
2003). Wild-type viruses expressing Vif are able to
prevent incorporation of APOBEC3G into the virion
by promoting its degradation in the cytoplasm of
the producer cell. Inhibition of APOBEC3G incor-
poration in the virus prevents hypermutation in
newly synthesized viral DNA following infection
of target cells due to C to U modifi cations during
minus stand DNA synthesis mediated by
APOBEC3G. Proteosome inhibitors interfere with
Vif dependent degradation of APOBEC3G
suggesting that these inhibitors can impede the
mechanisms used by the virus to evade the innate
defences of the host cell (Stopak et al. 2003; Sheehy
et al. 2003; Mehle et al. 2004; Yu et al. 2003).
Targeting an essential cellular process like the
proteosome is anticipated to be cytotoxic and not
well tolerated in vivo. Nevertheless, the highly
specifi c proteosome inhibitor epoxomicin, which
also inhibits HIV-1 maturation, is well tolerated in
mice (Meng et al. 1999). The proteosome inhibitor,
PS-341, is approved as a last resort treatment of
multiple myelomas and is associated with adverse
effects (Kane et al. 2006). The use of proteosome
inhibitors in HIV-1 infected individuals needs to
be considered in the context of the potential risk
benefi t and the net effect on inhibition of HIV-1
replication as proteosome inhibitors also enhance
the early step of the virus life-cycle by preventing
degradation of the reverse transcription complex
mediated by TRIM5α (Schwartz et al. 1998; Wu
et al. 2006; Wei et al. 2005).
Targeting HIV-1 Egress Mediated
by Vpu
The HIV-1 accessory protein, Vpu is a 16 kDa type 1
integral membrane protein that is indispensable
for viral pathogenesis (Li et al. 2005). Vpu plays
two distinctive roles in the viral life-cycle that
include down regulating host cell CD4 receptors
(Willey et al. 1992) and enhancing viral particle
release from the cell surface, the latter associated
with its ion channel forming properties (Schubert
et al. 1996a). Vpu is unique to HIV-1/SIVcpz
viruses (Binette and Cohen, 2004). Interestingly
the two closely related retroviruses HIV-2 and SIV,
which lack Vpu, are less pathogenic (Bour and
Strebel, 2003).
The role of Vpu in the viral budding process is
coupled to its ion channel forming properties,
which is predicted to be a pentameric structure
composed of fi ve transmembrane domains (Grice
et al. 1997). The Vpu ion channel is thought to
function by altering the electric potential at the
plasma membrane or alternatively by overcoming
host restriction factors for viral release (Neil et al.
2006). The HIV-1 Vpu is a member of viral ion
channel proteins called viroporins and is structur-
ally similar to the M2 ion channel protein of
infl uenza (Gonzalez and Carrasco, 2003; Hout et al.
2006). An interesting feature of Vpu is its role in
174
Wapling et al
Drug Target Insights 2007: 2
viral particle release from nondividing cells such
as macrophages (Deora and Ratner, 2001). In this
regard the rate of host cell proliferation is a deter-
mining factor for Vpu mediated viral particle
release (Deora and Ratner, 2001).
Analogues of amiloride (a sodium channel
blocker) inhibit Vpu ion channel activity. The
amiloride analogues, 5-(N,N-hexamethylene)
amiloride (HMA) and 5-(N,N-dimethyl)amiloride
(DMA) inhibit Vpu mediated virus budding and
viral replication in macrophages (Ewart et al. 2002;
Ewart et al. 2004). The inhibitory effects are
observed in the absence of cytotoxicity. Both
analogs exhibit strong inhibition of HIV-1 replica-
tion as measured by viral p24 levels in culture
supernatants (Ewart et al. 2004). HMA at 4 µM
suppresses viral p24 in culture supernatants to unde-
tectable levels for more than 10 days in culture
(Ewart et al. 2004). While amiloride analogues
demonstrate activity in macrophages, they fail to
inhibit HIV-1 replication in T-cells (Ewart et al.
2002). Nevertheless, Vpu ion channel inhibitors
have the potential for use in combination therapy,
targeting viral reservoirs and drug-resistant variants.
An amiloride derivative, BIT225, is currently being
pursued for drug development (Biotron Limited,
Sydney, NSW, Australia). BIT225 represents a
promising antiretroviral therapeutic although the
evaluation of its in vivo effi cacy will present a
challenge since inhibition of HIV-1 replication
appears to be restricted to nonproliferating cells.
Recent studies also suggest that rimantadine,
an ion channel blocker of infl uenza A viruses, can
be a useful lead compound for designing Vpu
inhibitors. Rimantadine and amantadine belong to
class of polycyclic amines that are active against
the M2 ion channel of infl uenza A but not against
HIV-1 Vpu (Hout et al. 2006). Studies indicate that
mutating histidine at residue 19 to alanine results
in a rimantadine sensitive Vpu ion channel demon-
strating the potential of this class of inhibitor as
HIV-1 ion channel blockers (Hout et al. 2006).
Vpu is also implicated to interact with certain
host cell restriction factors that interfere with viral
particle egress. The host cell protein, Vpu-binding
protein (UBP), is suggested to be a negative factor
for virus assembly (Callahan et al. 1998; Bour
and Strebel, 2003). UBP is a 34-kDa protein
that exhibits competitive binding with Vpu
and Gag (Callahan et al. 1998; Handley et al. 2001).
Overexpression of UBP has been reported to
significantly suppress viral particle release
suggesting that UBP is a negative factor that
requires displacement by Gag or Vpu (Callahan
et al. 1998). It is suggested that Vpu mediates its
effect on viral egress by facilitating membrane
targeting of Gag precursors (Handley et al. 2001;
Harila et al. 2006; Neil et al. 2006). Supporting
this notion, Vpu-defective particles appear in
internal membrane-bound compartments suggesting
a Gag targeting defect (Klimkait et al. 1990).
Another host cell restriction factor implicated
in viral release is an acid-sensitive potassium
channel-forming protein, TASK-1, which is down
regulated during viral infection (Hsu et al. 2004).
Due to the structural homology of TASK-1 and
HIV-1 Vpu, TASK-1 has been suggested to form
hetero-oligomers with Vpu which interferes with
both TASK-1 mediated conductance and Vpu ion
channel function (Hsu et al. 2004). Further delinea-
tion of the how these host cell factors interact with
Vpu is required in order to design small molecule
inhibitors that inhibit viral particle release.
Targeting HIV-1 Egress Mediated
by the p6 Late Domain
In the recent years, major advances have been made
in our understanding of how HIV-1 and other
retroviruses are released from infected cells. In the
early 1990s it was reported that mutations in the
p6 late domain inhibit virion particle release
(Gottlinger et al. 1991). Moreover, a PTAP sequence,
encompassing amino acids 7 to 10 of p6, is critical
for virus particle production (Huang et al. 1995).
The PTAP motif binds specifi cally to the host cell
protein, tumor suppressor gene 101 (TSG101),
resulting in the recruitment of components of the
endosomal sorting complex required for transport-I
(ESCRT-I) (VerPlank et al. 2001; Martin-Serrano
et al. 2001; Garrus et al. 2001; Demirov et al. 2002).
Deletion of the PTAP motif results in approximately
80% reduction in HIV-1 particle release. Similarly,
overexpression and silencing of TSG101 abolish
viral egress (Garrus et al. 2001; Demirov et al.
2002; Goila-Gaur et al. 2003).
NMR structure analysis of a 14 amino acid
peptide derived from p6 encompassing the PTAP
motif complexed with the UEV domain of TSG101
has revealed that the PTAP motif binds to a groove
in TSG101. This binding creates two main pockets:
the “A-P” pocket through contact of amino acids
7–10 and the “P” pocket through the binding of
amino acids 9–10 of the peptide (Pornillos et al.
175
Targeting the late stages of HIV-1 replication
Drug Target Insights 2007: 2
2002a; Pornillos et al. 2002b). Interruption of
this viral host protein-protein interaction with
compounds that bind to this pocket in TSG101 and
compete with Gag would potentially inhibit viral
particle release and the rate of cell-cell HIV-1
transmission (Bieniasz, 2006).
So far there has been one report that describes the
synthesis and selection of small molecules with up to
fi ve fold better binding capacity than a peptide that
contains the sequence of the wild type PTAP motif in
the L domain of HIV-1 (Liu et al. 2006). In this study,
the authors describe an approach previously employed
to obtain peptoids in which a key proline residue is
substituted with glycine at the N-terminus of the parent
Pro-rich sequence in order to improve binding
specifi city to Src homology 3 (SH3) domains (Nguyen
et al. 2000). Similarly, the proline rich PTAP domain
in p6 was considered a good candidate for the
N-substituted glycine residue approach. In this study,
the Tsg101 binding affi nity (Kd) of the 9-mer wild-type
PTAP containing peptide was 50 µM (Liu et al. 2006).
Binding constants for the highest affi nity peptoid-
hydrazones (designated 11q and 11p) were 17.5 and
9.8 µM, respectively. The highest affi nity peptoid
hydrazone was found to be the n-butyl-containing 11p
peptide, with a fi ve-fold increase in binding affi nity
compared to the wild-type 9-mer PTAP peptide. The
capacity of these peptides to compete with HIV-1 Gag
for TSG101 binding and their effect on HIV-1 egress
in vitro and in vivo remains to be determined.
Although disruption of viral-host cell interac-
tions is a very attractive approach to abolish virion
release and infectivity, interfering with the host cell
machinery could have major consequences for the
host (Bieniasz, 2006). TSG101 is a multifunctional
protein and plays a critical role in cell proliferation
as shown by studies conducted in TSG101 defi cient
mice (Ruland et al. 2001). TSG101 is involved in
cellular transcription and plays a central role in
endosomal sorting of cargo protein that is destined
for degradation by the proteasome. TSG101 is
recruited to the endosomal sorting pathway by a
specifi c interaction with the host cell protein, hepa-
tocyte growth factor-regulated tyrosine kinase
substrate (Hrs), which binds to TSG101 through a
PSAP motif (Lu et al. 2003). Disruption of this
interaction inhibits delivery of epidermal growth
factor receptor (EGFR) to the late endosomes. In
recruiting TSG101, it is believed that HIV-1 mimics
Hrs in order to enter the endosomal sorting pathway
and negotiate its release from the infected cell
(Pornillos et al. 2003). Thus, targeting TSG101
would result in the accumulation of proteins at the
plasma membrane and the disruption of protein
sorting within the infected cell. Specifi c inhibition
of HIV-1 budding by targeting the late domain
binding site on TSG101 will require preferential
inhibition of p6 binding compared to Hrs.
Conclusion
Considerable progress has been made in under-
standing the late steps of the viral life-cycle leading
to the production of infectious viral particles. Many
of these processes rely on protein:protein interac-
tions either between viral proteins or viral proteins
and host cell factors. The interactions between the
host and viral proteins have either a role in facili-
tating virus replication, as is observed for the viral
p6 and Tsg101, or are necessary to overcome nega-
tive effects of the host on virus replication, as
mediated by Vif. HIV-1 also relies on posttransla-
tional modifi cations mediated by the host cell
machinery in order for viral polyproteins to be
traffi cked to the appropriate compartment of the
cell for viral assembly and budding. Arguably, one
of the most effective drugs used to treat HIV-1
infected individuals are the HIV-1 PR inhibitors
that block viral maturation. This underscores
the effectiveness of targeting the late stage of
virus replication. Nevertheless, while some of the
strategies described in this review inhibit virus
replication in cell culture assays, they require
improvements in potency, specifi city and delivery
before being viable for use in the clinic.
Acknowledgements
J.W. is supported by an Australian Postgraduate
Award, S.S. and M. S-X are supported by funding
from the Australian Centre for HIV and Hepatitis
Virology Research and G.T. is supported by
funding from NHMRC R.D. Wright Biomedical
Career Development grant 235102 and NHMRC
Project Grant 381703.
Johanna Wapling and Seema Srivastava made
equal contribution to the review.
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/TransferFunctionInfo /Apply
/UCRandBGInfo /Preserve
/UsePrologue false
/ColorSettingsFile ()
/AlwaysEmbed [ true
]
/NeverEmbed [ true
]
/AntiAliasColorImages false
/DownsampleColorImages true
/ColorImageDownsampleType /Bicubic
/ColorImageResolution 300
/ColorImageDepth -1
/ColorImageDownsampleThreshold 1.50000
/EncodeColorImages true
/ColorImageFilter /DCTEncode
/AutoFilterColorImages true
/ColorImageAutoFilterStrategy /JPEG
/ColorACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/ColorImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000ColorACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000ColorImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasGrayImages false
/DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic
/GrayImageResolution 300
/GrayImageDepth -1
/GrayImageDownsampleThreshold 1.50000
/EncodeGrayImages true
/GrayImageFilter /DCTEncode
/AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG
/GrayACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/GrayImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000GrayACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000GrayImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasMonoImages false
/DownsampleMonoImages true
/MonoImageDownsampleType /Bicubic
/MonoImageResolution 1200
/MonoImageDepth -1
/MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true
/MonoImageFilter /CCITTFaxEncode
/MonoImageDict <<
/K -1
>>
/AllowPSXObjects false
/PDFX1aCheck false
/PDFX3Check false
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/PDFXNoTrimBoxError true
/PDFXTrimBoxToMediaBoxOffset [
0.00000
0.00000
0.00000
0.00000
]
/PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [
0.00000
0.00000
0.00000
0.00000
]
/PDFXOutputIntentProfile ()
/PDFXOutputCondition ()
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>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice