Pseudotyped Viruses: A Useful Platform for Pre-Clinical Studies Conducted in a BSL-2 Laboratory Setting

Abstract

The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus-cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses.

Keywords: SARS-CoV-2; in vitro model; neutralizing antibodies; pseudoviral particles; viral infection.

Figure 2

Schemes of the original VSV genome and the packaging system based on it. (A) Schematic representation of a single-stranded negative-sense RNA genome consisting of five primary genes coding for: N protein—nucleoprotein; P protein—phosphoprotein; M protein—matrix protein; G protein—glycoprotein; L protein [38]. (B) Recombinant packaging system. Step 1: Generation of rVSV-ΔG-envelope-transgene, where the envelope encodes the surface glycoprotein of the virus of interest and the transgene encodes the reporter protein. Step 2: Co-transfection with the obtained plasmid and VSV-system helper plasmids, resulting in rVSV-ΔG-surface glycoprotein of the virus of interest-reporter protein production [47,48].

Figure 1

Schemes of original HIV genome and lentiviral packaging systems based on it. (A) Schematic representation of HIV-1 original genome: LTR—long terminal repeat essential for viral transcription, reverse transcription, and integration; gag—gene encoding for proteins p24 (formation of the capsid), p17 (formation of the inner membrane layer), p7 (formation of the nucleoprotein/RNA complex), p6 (virus particle release); pol—gene encoding a set of enzymes including p10 (proteolytic cleavage of precursor proteins), p51 (transcription of HIV-1 RNA in proviral DNA), p15 (degradation of viral RNA in the viral RNA/DNA replication complex), p32 (integration of proviral DNA into the host genome); env—gene encoding for surface glycoprotein gp120 (attachment to the target cell), transmembrane protein gp41 (anchorage of gp120, fusion with the target cell); tat—gene encoding for regulatory protein p14 (activator of transcription of viral genes); rev—gene encoding for RNA splicing-regulator protein p19 (regulation of the export of non-spliced and partially spliced viral mRNA); vif—gene encoding for viral infectivity protein p23 ; vpr—gene encoding for virus protein r, or p15 (component of virus particles, interaction with p6); vpu—gene encoding for virus protein unique, or p16 (efficient virus particle release, control of CD4 degradation); nef—gene encoding for negative regulating factor, or p27 (influence on HIV replication, enhancement of infectivity, downregulation of CD4 on target cells). (B) Recombinant packaging systems comprising second and third generations of lentiviral vectors: Ψ—psi viral packaging signal sequence (packaging and delivering of transgene mRNA); RRE—cis-acting Rev response element (export from the nucleus to the cytoplasm of viral transcripts); cPPT—central polypurine tract (improvement of the vector integration and transduction efficiency); polyA—(promotion of transcript longevity); env—gene coding for glycoprotein of the virus of interest (replacement of the native lentiviral Env glycoprotein, providing vectors to transduce a particular set of cells); ΔU3—self-inactivating 3′LTR (viral transcription, reverse transcription and integration; contains a safety measure to prevent viral replication).