In vitro evolution of high-titer, virus-like vesicles containing a single structural protein Nina F. Rosea, Linda Buonocorea, John B. Schella, Anasuya Chattopadhyaya, Kapil Bahla, Xinran Liub, and John K. Rosea,1 aDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510; and bCenter for Cellular and Molecular Imaging, Yale University School of Medicine, New Haven, CT 06510 Edited* by Robert A. Lamb, Northwestern University, Evanston, IL, and approved October 22, 2014 (received for review August 6, 2014) Self-propagating, infectious, virus-like vesicles (VLVs) are gener- RNA. These proteins form a complex that directs replication of ated when an alphavirus RNA replicon expresses the vesicular the genomic RNA to form antigenomic RNA, which is then stomatitis virus glycoprotein (VSV G) as the only structural protein. copied to form full-length positive strand RNA and a subgenomic The mechanism that generates these VLVs lacking a capsid protein mRNA that encodes the structural proteins. The capsid protein has remained a mystery for over 20 years. We present evidence encases the genomic RNA in the cytoplasm and then buds from that VLVs arise from membrane-enveloped RNA replication facto- the cell surface in a membrane containing the SFV glycoproteins. ries (spherules) containing VSV G protein that are largely trapped Alphavirus RNA replication occurs inside light-bulb shaped, on the cell surface. After extensive passaging, VLVs evolve to membrane-bound compartments called spherules that initially grow to high titers through acquisition of multiple point muta- form on the cell surface and are then endocytosed to form cyto- tions in their nonstructural replicase proteins. We reconstituted pathic vacuoles containing multiple spherules (9). The replicase these mutations into a plasmid-based system from which high- proteins appear localized near the cytoplasmic side of the spher- ules (10). Positive-strand genomic RNA produced in the spherules titer VLVs can be recovered. One of these mutations generates is packaged into nucleocapsids before SFV budding. Alphavirus a late domain motif (PTAP) that is critical for high-titer VLV RNA replicons lacking structural protein genes can replicate ef- production. We propose a model in which the VLVs have evolved ficiently inside a cell, but they are incapable of propagating beyond in vitro to exploit a cellular budding pathway that is hijacked by the cell. many enveloped viruses, allowing them to bud efficiently from the We have been interested in developing VLVs as a vaccine cell surface. Our results suggest a basic mechanism of propagation platform (5, 6). However, the relatively low titers generated were MICROBIOLOGY that may have been used by primitive RNA viruses lacking capsid a major limitation of the system. We undertook the extensive proteins. Capsids may have evolved later to allow more efficient serial passaging studies described here to determine whether packaging of RNA, greater virus stability, and evasion of innate VLVs could evolve in culture to grow to high titers. We succeeded immunity. in generating VLVs that grow to at least 1,000-fold higher titers. In the process of studying these VLVs, we generated data sug- VSV glycoprotein | evolution | SFV replicon | late domain gesting the mechanism of VLV formation. In addition, our data suggest that the high-titer VLVs have evolved through passaging nveloped RNA viruses have highly organized structures. One to use a cellular budding machinery that is exploited by many Eor more capsid proteins encase their RNA, matrix proteins enveloped viruses to drive efficient budding. often lie between the capsid and the membrane, and one or more Many enveloped RNA viruses use components of a cellular vesicular budding machinery to drive efficient budding from the transmembrane glycoproteins can interact with the matrix or cell surface (11–13). Short sequence motifs called late domains capsid proteins to direct efficient particle assembly (1). Once the in their structural proteins recruit cellular protein complexes particles are released from cells, one or more glycoproteins in called ESCRT (endosomal sorting complex required for trans- the viral envelope bind cellular receptors and catalyze membrane port). The ESCRT complexes are normally involved in budding fusion to allow the viruses to enter new cells (2). Vesicular stomatitis virus (VSV) is a negative-strand RNA virus Significance that encodes a single membrane glycoprotein (G), a matrix protein, and a nucleocapsid protein as well as two proteins that form the viral polymerase (3). Remarkably, when cells are transfected with All known membrane-enveloped RNA viruses have capsid an alphavirus RNA replicon encoding only the alphavirus non- proteins that encase their RNA genomes. This paper shows that structural replicase proteins and the VSV G protein, infectious, infectious, membrane-enveloped, virus-like vesicles with RNA self-propagating membrane-enveloped vesicles containing the VSV genomes can evolve in vitro to grow to high titers without G protein are generated (4). These infectious, virus-like vesicles a capsid protein. The infectious vesicles are apparently gener- (VLVs) grow to only low titers of 104 to 105 infectious units (i.u.) ated from RNA replication factories called spherules that bud per mL, but propagate like a virus in tissue culture cells. The from the cell surface. They evolve in vitro to bud with high vesicles contain the genomic RNA and VSV G protein, but unlike efficiency through the acquisition of multiple mutations in the known enveloped RNA viruses, they lack a capsid protein encasing non-structural replicase proteins. One mutation generates their RNA resulting in a low buoyant density (4). The mechanism a critical motif found in many viral structural proteins. This by which these VLVs are generated has never been determined. A motif is involved in recruiting cellular machinery to drive effi- nonspecific packaging was postulated because the VLVs contained cient budding. Prior to the evolution of capsid proteins, prim- both genomic RNA and subgenomic mRNA (4) and the titers itive RNA viruses may have used this budding mechanism. could be increased by sonication of the cells. Despite the low titers, VLVs expressing other proteins have proven useful as experi- Author contributions: N.F.R. and J.K.R. designed research; N.F.R., L.B., J.B.S., A.C., K.B., X.L., mental vaccines (5, 6). and J.K.R. performed research; N.F.R., L.B., J.B.S., and J.K.R. analyzed data; and N.F.R., L.B., The alphavirus replicon used in the studies described above and J.K.R. wrote the paper. was derived from Semliki Forest Virus (SFV), a positive-strand, The authors declare no conflict of interest. membrane-enveloped RNA virus that encodes four nonstructural *This Direct Submission article had a prearranged editor. proteins called nsP 1–4 and three structural proteins: capsid, and 1To whom correspondence should be addressed. Email: [email protected]. – the E1 and E2 transmembrane glycoproteins (7, 8). The nsP 1 4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins are translated from the first two-thirds of the genomic 1073/pnas.1414991111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1414991111 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 of vesicles into cellular multivesicular bodies (MVB) as well as We compared the growth rate of the VLVs to VSV using tra- other cellular processes (12). Retroviruses, paramyxoviruses, filo- ditional one-step growth curves and found that the maximal VLV viruses, and rhabdoviruses have been shown to use components of titers (∼108 pfu/mL) were typically reached by between 12 and 24 h the ESCRT pathway to drive budding (11–13). However, other after high multiplicity infection. In contrast, maximal VSV titers 9 enveloped RNA viruses including SFV and influenza virus do not (∼10 pfu/mL) were reached between 10 and 12 h after infection. use the ESCRT pathway in budding (14–16). Purified p50 VLVs Contain VSV G as Their Structural Protein. To de- Results termine what structural proteins were in the p50 VLVs, we × 6 Extensive Passaging Generates High-Titer VLVs. A plasmid with an infected 2 10 BHK cells with p50 VLVs or with VSV at a efficient promoter driving synthesis of SFV replicon RNA inside multiplicity of infection (MOI) of 10. After 24 h when the cells showed extensive cytopathic effect (c.p.e.), the VSV particles or cells was described previously (17). After transfection of this DNA the VLVs in the medium were purified through two rounds of onto cells, the replicon RNA is synthesized in the nucleus, and ultracentrifugation. The proteins in the VLVs and VSV particles then moves to the cytoplasm where it is translated and begins the were then analyzed by SDS/PAGE followed by silver staining. As replication cycle. We previously constructed a derivative of this shown in Fig. 2A, VSV virions contained the five proteins N, P, M, vector with two subgenomic SFV mRNA promoters (6). The first G, and L, whereas the purified p50 VLVs showed VSV G protein promoter drives expression of VSV G mRNA and the second as the only apparent structural protein. There was no trace of the promoter can drive expression of mRNAs encoding other antigens. SFV capsid protein (29.8 kDa) consistent with the loss of its ex- The infectious VLVs derived from these constructs are potent pression during early passages. The total yield of VLV protein experimental vaccines, but they released relatively low titers of only from 5 × 106 BHK cells infected with the p50 VLVs was ∼20 μg, about 104 to 105 infectious units (i.u.)/mL These titers could be whereas the same number of cells infected with VSV yielded increased about 100-fold by sonicating the cells (6). about 60 μg of total viral protein. We did not recover measurable To determine whether VLVs might be able to evolve to grow to protein yields from medium derived from uninfected BHK cells.