In vitro evolution of high-titer, virus-like vesicles containing a single structural protein

Abstract

Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein.The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which hightiter VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity.VSV glycoprotein | evolution | SFV replicon | late domain

G gene-deficient single-round rabies viruses for neuronal circuit analysis

“…Moreover, as illustrated using G gene-deficient RABV (SAD G), M-driven budding and release of bald, spikeless virus can take place in the complete absence of G, although with a 30-fold reduction of particle production . Notably, the G protein of VSV can not only contribute to rhabdovirus budding (Robison and Whitt, 2000), but has autonomous budding activity, as illustrated in experiments in which VSV G was sufficient to mobilize alphavirus replicons, yielding a novel type of infectious vesicles (Rolls et al, 1994;Rose et al, 2014). The relative independence of rhabdovirion formation from their cognate G proteins provides the basis for successful pseudotyping with heterologous proteins.…”

Virus-Like Vesicle-Based Therapeutic Vaccine Vectors for Chronic Hepatitis B Virus Infection

“…Furthermore, VLVs demonstrate an ability to induce potent immune responses while lacking pathogenicity in mouse and nonhuman primate models (7,8). Recently, this VLV system has been evolved to reach high titers, further improving its potential as a vaccine vector while still lacking detectable pathogenicity (11). In this study, we used this high-titer evolved vector system to explore how VLVs expressing either the middle version of the HBV envelope protein (MHBs) or the HBV core protein (HBcAg) may be developed as a therapeutic vaccine for chronic HBV infection.…”

Infectious Chikungunya Virus (CHIKV) with a Complete Capsid Deletion: a New Approach for a CHIKV Vaccine

“…infectious microvesicles (iMVs) (37). Additionally, infectious viruslike vesicles (VLVs) were generated when cells were transfected with an SFV replicon without SFV structural proteins expressing only vesicular stomatitis virus glycoprotein (VSV-G) (38)(39)(40) or murine leukemia virus envelopes (41). The possible mechanism of VLV formation is vesiculation of the plasma membrane to generate particles that include VSV-G and SFV-G RNAs (38).…”