








key: cord-262752-bwofzbwa
authors: Li, Qianqian; Liu, Qiang; Huang, Weijin; Li, Xuguang; Wang, Youchun
title: Current status on the development of pseudoviruses for enveloped viruses
date: 2017-12-07
journal: Rev Med Virol
DOI: 10.1002/rmv.1963
sha: 
doc_id: 262752
cord_uid: bwofzbwa

Emerging and reemerging infectious diseases have a strong negative impact on public health. However, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. Alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. As pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. Importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. However, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. This review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields.

A pseudovirus is a recombinant viral particle with its core/backbone and envelope proteins derived from different viruses 1 ; moreover, the genes inside the pseudovirus are usually altered or modified so that they are unable to produce the surface protein on their own. As such, an additional plasmid or stable cell line expressing the surface proteins is needed to make the pseudovirus. 2 Pseudoviruses are capable of infecting susceptible cells, but they only replicate for 1 round in the infected host cells. 3 Compared with wild-type (WT) viruses, pseudoviruses can be safely handled in biosafety level (BSL)-2 laboratories 4 and are usually easier to manipulate experimentally. 5 Nevertheless, the conformational structure of pseudoviral surface proteins bears high similarity to that of the native viral proteins, and these surface proteins can effectively mediate viral entry into host cells. Therefore, pseudoviruses are widely used for the study of cellular tropism, 6 receptor recognition, 7 and virus inhibition, 8 as well as for developing and evaluating antibodies 9 and vaccines. 10 In addition, data from in vitro pseudovirus-based antiviral assays and in vivo biodistribution analyses have been found to correlate very well with the results generated by using live WT viruses. 11, 12 As pseudoviruses have usually been engineered to carry reporter genes, it is much easier to perform quantitative analyses on these viruses than on WT viruses, 13 and the number of pseudovirus-infected cells has been shown to be directly proportional to reporter gene expression. The reporter genes usually encode either an enzyme or a fluorescent protein, with each option having its particular strengths and weaknesses. Specifically, chemiluminescence assays usually have lower background and are more sensitive, but the data acquisition and analyses for these assays are time-consuming and more expensive.

In contrast, assays using a florescence protein, such as green fluorescent protein, are cheaper and easier to operate in both in vitro and in vivo systems; however, they are less sensitive and may have higher background. [14] [15] [16] In this review, we provide an update on the development and application of pseudoviral systems and discuss some challenging technical issues. 

The HIV-1 packaging system is the most widely used pseudovirus packaging system. To make this packaging system, HIV genes are selectively cloned into DNA vectors. Specifically, 2 to 4 plasmids are used as the vectors, a strategy that aims to minimize viral gene recombination and thereby reduce the possibility of reversion to the WT virus. Table 1 lists the currently used HIV-1-based systems.

The original 2-plasmid system comprises 1 envelope plasmid and 1 HIV-1 backbone plasmid, ie, pSG3Δenv and pNL4-3 (the env gene sequence in pSG3Δenv is destroyed). 51 However, this system is not ideal, as its viral yield is usually very low. Improvements have been made through the addition of other sequences for better reporter gene expression. Specifically, our research group inserted the reporting gene, firefly luciferase (Fluc), into pSG3Δenv between env and nef to produce pSG3Δenv.Fluc.Δnef. In addition, we also generated pSG3Δenv.CMVFluc, which carries a functional nef and CMV promoter to drive the reporter gene. 10 By using these optimized backbone and envelope protein expression plasmids, our group succeeded in producing several HIV pseudoviruses carrying the envelope proteins of EBOV, MARV, LASV, Middle East respiratory syndrome-coronavirus, rabies virus (RV), chikungunya virus, and nipah virus (NiV). The yields of pseudoviruses constructed with this optimized system were improved by 100 to 1000-folds as compared with those of pseudoviruses constructed with pNL4-3.Luc.R-E. 52

The HIV 3-plasmid system is usually comprised of a packaging plasmid, a transfer plasmid containing the reporter gene, and an envelope-expressing plasmid. Specifically, this system is made by splitting the HIV-1 backbone into separate packing and transfer plasmids. The packaging plasmid expresses the gag and pol proteins, while the transfer plasmid contains the cis-regulatory elements needed for HIV reverse transcription, integration, and packaging as well as multiple cloning sites and a reporter gene under the control of a heterogeneous promoter. 48-50 The envelope-expressing plasmid is made of a vector carrying the envelope gene driven by a CMV promoter.

The HIV 4-plasmid system is based on the 3-plasmid system, with the Rev protein being expressed by an additional, separate plasmid. Specifically, this system comprises 1 packaging plasmid expressing the gag and pol proteins, a second packaging plasmid encoding Rev, 1 plasmid producing the WT envelope protein, and a transfer plasmid with cis-regulatory elements. 53 These 3 HIV-based systems were reported by different groups, and, as yet, no comparison has been made among the different systems in safety and efficiency. Our group is able to drastically improve the viral yield with 2-plasmid system, while no safety issue of this HIV pseudoviral systems was observed in animals. 52 2.1.2 | The simian immunodeficiency virus packaging system HIV Env Cellular tropism, neutralization antibody assay, impact of Env amino acid mutation, and glycosylation on the neutralization epitope, drug screening and validation, and receptor recognition pSG3ΔEnv; pNL4-3.Luc.R-E- 

The VSV packaging system is a versatile tool for making pseudotyped viruses; this system is advantageous in that it has no stringent selectivity for the envelope proteins, and the resulting virus may be manipulated in a BSL-2 laboratory. Early studies jointly employed VSV and a second virus to coinfect cells, resulting in the pseudotyped virus carrying the core of VSV with envelope proteins derived from the other virus. 60 Stillman et al were the first to clone the VSV genome into a plasmid to make stable VSV, 61 which was subsequently used to generate pseudoviruses carrying heterogeneous glycoprotein. 62, 63 Various reporter genes were successively inserted into this plasmid to facilitate its easy detection. 64, 65 Some examples of VSV-based pseudovirus system are listed in Table 2 .

Notably, when the VSV packaging system is used to make a pseudovirus, there may be residual VSV virus mixed with the pseudovirus, thereby complicating the neutralization assay in which it is used or producing false-positive results. Preferably, the amount of VSV should be minimized; however, if excess VSV is believed to be interfering with a pseudovirus-based assay, treatment of the pseudovirus preparation with a VSV neutralizing antibody could be considered before its use in future assays.

The MLV packaging system, also called the retroviral system, is commonly used to make pseudoviruses. Table 3 lists the pseudoviruses packaged by the MLV system that have been reported in the literature.

Early work by Witte and colleagues showed that when they used VSV to infect the cells in which MLV is packaged, they were able to harvest pseudovirus for use in neutralization antibody assays. 98 Since then, the genome of MLV had been split into 2 parts: one encoding gag-pol and the other containing the reporter gene. The 2 gene sets were further cloned into plasmids to generate highly efficient MLV packaging systems. 99 To improve the stability of this system, investigators established several cell lines that were confirmed to be stable in transfection and expression. Murine leukemia virus may actually be a better choice than HIV as a packaging system in some cases. For example, in studying LASV-mediated entry into cells, Cosset et al compared the MLV and HIV systems and found that the former is 5-fold more efficient than the latter. 100 

The aforementioned pseudovirus packaging systems have not always been successful in generating certain types of pseudoviruses. In those cases, other alternatives such as reverse genetics have been reported.

For example, Hu et al prepared a pseudotyped dengue virus (DENV) types 1 to 4 by using the HIV system, but its titer was insufficiently high. 101 However, by using reverse genetics, reporter genes were 

A high yield is needed for practical applications of the pseudoviruses.

There are several factors that can critically influence their yield/titer.

In general, the subcellular localization for viral packaging and matura- successfully used the MLV packaging system to generate a pseudotyped influenza virus. 108 Cosset et al reached the same conclusion for pseudotyped LASV preparations. 100 Moreover, in our experiences, pseudotyped LASV packaged by the VSV system had a higher titer than that produced by using the HIV system. Furthermore, the VSV system was able to incorporate hantavirus glycoprotein that had failed to be packaged by HIV. Nonetheless, our group developed a modification of the HIV packaging system that could improve the pseudoviral yield by 100-fold; specifically, the backbone plasmid pSG3ΔEnv.CMVFluc that was developed in our lab was superior to pNL4-3.Luc.R-E-. 10 

The packaging conditions can also drastically influence the pseudovirus yield. In the HIV packaging system, the pseudovirus titer could be 

Pseudoviruses have been widely used for conducting in vitro studies on the interaction between the virus and the host cells. 112 They have also proven to be very useful for in vivo studies, particularly studies on the mechanism of viral infection as well as on the biodistribution. 113 Our lab used a pseudotyped RV carrying reporter genes to establish an in vivo imaging model in mice. This mouse model was used to study viral tissue tropism and its dynamic change over time. 10 We also established a pseudotyped EBOV mouse model; the EBOV pseudoviruses were mainly detected in the thymus and spleen following viral infection, revealing that the pseudotyped EBOV and WT EBOV have the same tissue tropism. 52 Other groups have also used pseudotyped HSV-1

and MARV in small animal models to investigate viral infections. 114 4.2 | Application of pseudoviral systems to neutralization antibody and antibody-dependent cellmediated cytotoxicity assay Antibody neutralization assay based on pseudoviruses has been widely used, particularly for the analyses of some virulent viruses that would otherwise need to be handled in BSL-3 or BSL-4 laboratories. Compared with the traditional assays, the reported pseudovirus-based assays have demonstrated a good correlation with the WT virus-based assay; the pseudovirus-based assays are usually high-throughput procedures with fewer amounts of serum samples needed. 1, 104, 115, 116 Wilkinson et al compared 22 platform technologies for assaying antibody against EBOV with neutralization assays by using the WT virus and found that the 5 best assays included methods based on WT and VSV pseudotype neutralization and ELISA, but the lentiviral and other platforms were problematic. 117 

No false start for novel pseudotyped vectors

Vesicular stomatitis virus pseudotypes of retroviruses

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system

Lassa and Ebola virus inhibitors identified using minigenome and recombinant virus reporter systems

High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses

Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

A comparative high-throughput screening protocol to identify entry inhibitors of enveloped viruses

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system

Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison

A safe and sensitive enterovirus A71 infection model based on human SCARB2 knock-in mice

Comparison of two high-throughput assays for quantification of adenovirus type 5 neutralizing antibodies in a population of donors in China

Development of a Moloney murine leukemia virus-based pseudotype anti-HIV assay suitable for accurate and rapid evaluation of HIV entry inhibitors

Characterization of retroviral and lentiviral vectors pseudotyped with xenotropic murine leukemia virus-related virus envelope glycoprotein

Development of a triple-color pseudovirion-based assay to detect neutralizing antibodies against human papillomavirus

Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation

Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes

Characterization of Chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in E1 glycoprotein

Development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection

Chikungunya virus glycoproteins pseudotype with lentiviral vectors and reveal a broad spectrum of cellular tropism

Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo

Toremifene interacts with and destabilizes the Ebola virus glycoprotein

Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses

In vitro evaluation of cyanovirin-N antiviral activity, by use of lentiviral vectors pseudotyped with filovirus envelope glycoproteins

Packaging HIV-or FIV-based lentivector expression constructs and transduction of VSV-G pseudotyped viral particles

Pseudotype formation between enveloped RNA and DNA viruses

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins

Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles

Generation of VSV pseudotypes using recombinant DeltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

Quantification of lyssavirus-neutralizing antibodies using vesicular stomatitis virus pseudotype particles

Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

A system for functional analysis of Ebola virus glycoprotein

Rho GTPases modulate entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors

Characterization of pseudotype VSV possessing HCV envelope proteins

Use of vesicular stomatitis virus pseudotypes bearing Hantaan or Seoul virus envelope proteins in a rapid and safe neutralization test

A pseudotype vesicular stomatitis virus containing Hantaan virus envelope glycoproteins G1 and G2 as an alternative to hantavirus vaccine in mice

Study of Andes virus entry and neutralization using a pseudovirion system

Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies

Analyses of entry mechanisms of novel emerging viruses using pseudotype VSV system

Characterization of the interaction of Lassa fever virus with its cellular receptor alpha-dystroglycan

Analysis of Lujo virus cell entry using pseudotype vesicular stomatitis virus

Development of a neutralization assay for Nipah virus using pseudotype particles

EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus

A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein

Involvement of ceramide in the propagation of Japanese encephalitis virus

Preparation of vesicular stomatitis virus pseudotype with Chikungunya virus envelope protein

Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

A vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza A virus

Analysis of the entry mechanism of Crimean-Congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system

Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines

Murine leukemia virus pseudotypes of La Crosse and Hantaan Bunyaviruses: a system for analysis of cell tropism

Truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles

Assessment of HIV-1 entry inhibitors by MLV/HIV-1 pseudotyped vectors

Functional murine leukemia virus vectors pseudotyped with the visna virus envelope show expanded visna virus cell tropism

Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production

Coreceptor switch of [MLV(SIVagm)] pseudotype vectors by V3-loop exchange

Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein

Murine leukemia virus (MLV)-based coronavirus spike-pseudotyped particle production and infection

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus

Transduction of Schistosoma Japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes

Establishment of retroviral pseudotypes with influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and specific detection of neutralizing antibodies

Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus

A transient three-plasmid expression system for the production of high titer retroviral vectors

Characterization of Lassa virus cell entry and neutralization with Lassa virus pseudoparticles

Characterization of retrovirusbased reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses

A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

A high-throughput assay using dengue-1 virus-like particles for drug discovery

Pseudotypes: your flexible friends

A plasma membrane localization signal in the HIV-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into VSV virions

Truncation of the enzootic nasal tumor virus envelope protein cytoplasmic tail increases Env-mediated fusion and infectivity

Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling

A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies

Optimized large-scale production of high titer lentivirus vector pseudotypes

A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes

The optimisation of pseudotyped viruses for the characterisation of immune responses to equine influenza virus

Pseudotyping viral vectors with emerging virus envelope proteins

Applications of bioluminescence imaging to the study of infectious diseases

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

The use of pseudotypes to study viruses, virus sero-epidemiology and vaccination

Optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China

Comparison of platform technologies for assaying antibody to Ebola virus

Novel cross-reactive monoclonal antibodies against Ebola virus glycoproteins show protection in a murine challenge model

High-throughput screening of viral entry inhibitors using pseudotyped virus

Teicoplanin inhibits Ebola pseudovirus infection in cell culture

Identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay

A systematic screen of FDAapproved drugs for inhibitors of biological threat agents

Characterization of the inhibitory effect of an extract of Prunella vulgaris on Ebola virus glycoprotein (GP)-mediated virus entry and infection

Properties of wild-type, C-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral vector particles

Cholesterol supplementation during production increases the infectivity of retroviral and Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G)

Short communication: potential risk of replication-competent virus in HIV-1 Env-pseudotyped virus preparations

Current status on the development of pseudoviruses for enveloped viruses

The authors have no competing interest.

