Significance
All known membrane-enveloped RNA viruses have capsid proteins that encase their RNA genomes. This paper shows that infectious, membrane-enveloped, virus-like vesicles with RNA genomes can evolve in vitro to grow to high titers without a capsid protein. The infectious vesicles are apparently generated from RNA replication factories called spherules that bud from the cell surface. They evolve in vitro to bud with high efficiency through the acquisition of multiple mutations in the non-structural replicase proteins. One mutation generates a critical motif found in many viral structural proteins. This motif is involved in recruiting cellular machinery to drive efficient budding. Prior to the evolution of capsid proteins, primitive RNA viruses may have used this budding mechanism.
Keywords: VSV glycoprotein, evolution, SFV replicon, late domain
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 high-titer 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.
Enveloped RNA viruses have highly organized structures. One or more capsid proteins encase their RNA, matrix proteins often lie between the capsid and the membrane, and one or more transmembrane glycoproteins can interact with the matrix or capsid proteins to direct efficient particle assembly (1). Once the particles are released from cells, one or more glycoproteins in the viral envelope bind cellular receptors and catalyze membrane fusion to allow the viruses to enter new cells (2).
Vesicular stomatitis virus (VSV) is a negative-strand RNA virus 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 an alphavirus RNA replicon encoding only the alphavirus nonstructural replicase proteins and the VSV G protein, infectious, self-propagating membrane-enveloped vesicles containing the VSV G protein are generated (4). These infectious, virus-like vesicles (VLVs) grow to only low titers of 104 to 105 infectious units (i.u.) per mL, but propagate like a virus in tissue culture cells. The vesicles contain the genomic RNA and VSV G protein, but unlike known enveloped RNA viruses, they lack a capsid protein encasing their RNA resulting in a low buoyant density (4). The mechanism by which these VLVs are generated has never been determined. A nonspecific packaging was postulated because the VLVs contained both genomic RNA and subgenomic mRNA (4) and the titers could be increased by sonication of the cells. Despite the low titers, VLVs expressing other proteins have proven useful as experimental vaccines (5, 6).
The alphavirus replicon used in the studies described above was derived from Semliki Forest Virus (SFV), a positive-strand, membrane-enveloped RNA virus that encodes four nonstructural proteins called nsP 1–4 and three structural proteins: capsid, and the E1 and E2 transmembrane glycoproteins (7, 8). The nsP 1–4 proteins are translated from the first two-thirds of the genomic RNA. These proteins form a complex that directs replication of the genomic RNA to form antigenomic RNA, which is then copied to form full-length positive strand RNA and a subgenomic mRNA that encodes the structural proteins. The capsid protein encases the genomic RNA in the cytoplasm and then buds from the cell surface in a membrane containing the SFV glycoproteins. Alphavirus RNA replication occurs inside light-bulb shaped, membrane-bound compartments called spherules that initially form on the cell surface and are then endocytosed to form cytopathic vacuoles containing multiple spherules (9). The replicase proteins appear localized near the cytoplasmic side of the spherules (10). Positive-strand genomic RNA produced in the spherules is packaged into nucleocapsids before SFV budding. Alphavirus RNA replicons lacking structural protein genes can replicate efficiently inside a cell, but they are incapable of propagating beyond the cell.
We have been interested in developing VLVs as a vaccine platform (5, 6). However, the relatively low titers generated were a major limitation of the system. We undertook the extensive serial passaging studies described here to determine whether VLVs could evolve in culture to grow to high titers. We succeeded in generating VLVs that grow to at least 1,000-fold higher titers. In the process of studying these VLVs, we generated data suggesting the mechanism of VLV formation. In addition, our data suggest that the high-titer VLVs have evolved through passaging to use a cellular budding machinery that is exploited by many enveloped viruses to drive efficient budding.
Many enveloped RNA viruses use components of a cellular vesicular budding machinery to drive efficient budding from the cell surface (11–13). Short sequence motifs called late domains in their structural proteins recruit cellular protein complexes called ESCRT (endosomal sorting complex required for transport). The ESCRT complexes are normally involved in budding of vesicles into cellular multivesicular bodies (MVB) as well as other cellular processes (12). Retroviruses, paramyxoviruses, filoviruses, and rhabdoviruses have been shown to use components of the ESCRT pathway to drive budding (11–13). However, other enveloped RNA viruses including SFV and influenza virus do not use the ESCRT pathway in budding (14–16).
Results
Extensive Passaging Generates High-Titer VLVs.
A plasmid with an efficient promoter driving synthesis of SFV replicon RNA inside cells was described previously (17). After transfection of this DNA onto cells, the replicon RNA is synthesized in the nucleus, and then moves to the cytoplasm where it is translated and begins the replication cycle. We previously constructed a derivative of this vector with two subgenomic SFV mRNA promoters (6). The first promoter drives expression of VSV G mRNA and the second promoter can drive expression of mRNAs encoding other antigens. The infectious VLVs derived from these constructs are potent experimental vaccines, but they released relatively low titers of only about 104 to 105 infectious units (i.u.)/mL These titers could be increased about 100-fold by sonicating the cells (6).
To determine whether VLVs might be able to evolve to grow to high titers through passaging in tissue culture, we began with the DNA construct diagramed in Fig. 1. The SFV replicon RNA generated by this construct expresses VSV G from the first subgenomic promoter and the SFV capsid protein from the second promoter. We derived infectious VLVs from this construct after transfection of BHK cells and found low titers typical of VLVs (∼105 i.u./mL). We then continued passaging of the VLVs on BHK cells over a period of one year. In the initial passages we transferred 5% of the medium from cells showing cytopathic effect (CPE) onto fresh BHK cells on 6 cm dishes, and strong CPE typically developed in 3–5 d. Expression of the SFV capsid protein was completely lost by passage eight suggesting that its expression was inhibiting particle production. With continued passaging we noted increasing VLV titers and a much more rapid CPE developing in less than 24 h. By passage fifty we found that the VLVs had evolved to the point where they formed plaques on BHK cells within two days and attained titers of >5 × 107 plaque forming units (pfu)/mL At this point we picked a large VLV plaque and used this to generate a cloned VLV stock that we call p50 VLV. This p50 VLV grows to titers of ∼108 pfu/mL, an increase of ∼1000-fold over the starting vector.
Fig. 1.
Large deletion and eleven amino acid changes occurred in high-titer evolved VLVs. A diagram of the plasmid pCMV-SFVG-CAP) used to derive VLVs for passaging is shown. The indicated promoter (CMV) drives expression of the positive strand replicon RNA encoding the nonstructural proteins. The subgenomic SFV mRNA promoters (small green arrows) drive expression of two mRNAs encoding VSV G or the SFV capsid protein from the full-length antigenomic RNA. After 50 passages of VLVs the consensus sequence of p50 VLV RNA derived from a single plaque showed the large deletion illustrated removing the second SFV promoter and the capsid gene. The x's indicate the approximate positions of the 11 nucleotide changes that change amino acids in nsP proteins and in VSV G.
We compared the growth rate of the VLVs to VSV using traditional one-step growth curves and found that the maximal VLV titers (∼108 pfu/mL) were typically reached by between 12 and 24 h after high multiplicity infection. In contrast, maximal VSV titers (∼109 pfu/mL) were reached between 10 and 12 h after infection.
Purified p50 VLVs Contain VSV G as Their Structural Protein.
To determine what structural proteins were in the p50 VLVs, we infected 2 × 106 BHK cells with p50 VLVs or with VSV at a multiplicity of infection (MOI) of 10. After 24 h when the cells showed extensive cytopathic effect (c.p.e.), the VSV particles or the VLVs in the medium were purified through two rounds of ultracentrifugation. The proteins in the VLVs and VSV particles were then analyzed by SDS/PAGE followed by silver staining. As shown in Fig. 2A, VSV virions contained the five proteins N, P, M, G, and L, whereas the purified p50 VLVs showed VSV G protein as the only apparent structural protein. There was no trace of the SFV capsid protein (29.8 kDa) consistent with the loss of its expression during early passages. The total yield of VLV protein from 5 × 106 BHK cells infected with the p50 VLVs was ∼20 μg, whereas the same number of cells infected with VSV yielded about 60 μg of total viral protein. We did not recover measurable protein yields from medium derived from uninfected BHK cells.
Fig. 2.
Characterization of p50 VLVs. (A) Photograph showing SDS/PAGE analysis of VSV virions and p50 VLVs. Purified VSV virions or p50 VLVs (1 μg of protein each) were fractionated by SDS-10% PAGE (Invitrogen) and the proteins bands were stained and developed using a Pierce Silver Stain kit. The positions and molecular weights of the VSV structural proteins N, P, M, G, and L are indicated. (B and C) TEM images of fixed and stained VLVs showing spikes protruding from the vesicle membrane. (D) TEM image of fixed and stained VLV apparently releasing nucleic acid. (E) TEM image of large field of unfixed and purified VLVs labeled with an anti-VSV G mouse monoclonal antibody (I1) and a secondary anti-mouse antibody conjugated with 12nm gold particles. Scale bars for all TEM images are as indicated.
Characterization of the p50 VLVs by Electron Microscopy.
The purified VLVs used for the protein characterization were first imaged by transmission electron microscopy (TEM) following glutaraldehyde fixation and negative staining with uranyl acetate. Examples are shown in Fig. 2 B–D. Many of the vesicles appeared to have protein spikes on their surfaces (Fig. 2 B and C, arrows). Some of the vesicles prepared for TEM by this method also appeared to be broken and releasing nucleic acid (Fig. 2D).
We next performed TEM on VLVs that were unfixed, and labeled with antibody to VSV G followed by a secondary antibody conjugated to 12-nm gold particles. The vesicles were then negative-stained with phosphotungstic acid (Fig. 2E). The TEM image shows clear surface labeling of numerous vesicles with a halo of gold particles indicating the presence of VSV G on their surfaces. Vesicle diameters were in the 50–200 nm range. The size distribution was similar to that observed for the low titer VLVs described previously (4).
Sequence of the p50 VLV Genome and Reconstruction into a Plasmid DNA.
To determine a consensus sequence of the p50 VLV genome we performed reverse transcription and PCR to generate overlapping DNA fragments covering the entire genome, and sequenced these fragments. The assembled sequence revealed a large deletion of 1,672 nucleotides (Fig. 1), which removed the second SFV promoter, the entire capsid gene, and all except 187 nucleotides of SFV sequences preceding the poly(A). In addition to this deletion, there were 16 single-base changes (Table 1). Ten of these changed amino acids in all four of the SFV nonstructural proteins, one changed an amino acid in VSV G, and four mutations were silent (Fig. 1 and Table 1).
Table 1.
Nucleotide and amino acid changes in p50 VLVs
| Nucleotide change | Amino acid change and context* | Protein affected |
| G-4700-A | G-106-E (AASEKVL) | nsP1 |
| A-5424-G | None | |
| G-5434A | V-351-I (ATDITPE) | nsP1 |
| T-5825-C | L-481-S (KRESIPV) | nsP1 |
| T-5930-C | I-516-T (LVPTAPA) | nsP1 |
| A-6047-G | D-555-G (QPNGVLL) | nsP2 |
| G-6783-A | None | |
| G-6963-A | None | |
| G-7834-A | A-1151-T (ALVTEYK) | nsP2 |
| T-8859-A | None | |
| T-8864-C | M-1494-T (AIDTRTA) | nsP3 |
| G-9211-A | A-1610-T (ERITRLR) | nsP3 |
| A-10427-G | N-2015-S (TLQSVLA) | nsP4 |
| G-11560-A | E-2393-K (SRYKVEG) | nsP4 |
| A-11871-G | N-34-D (NWHDDLI) | VSV G |
| T-11978-C | None |
Amino acids are numbered in the SFV nsP1-4 polyprotein, or in the VSV G protein. Bold underlined text indicates the amino acid that was changed.
To reconstitute high-titer p50 VLV sequence into a plasmid DNA and begin to analyze the critical sequence changes, we began a stepwise reconstruction (Fig. 3). First we introduced the 1,672-nucleotide deletion into the plasmid using a synthetic DNA fragment containing the deletion. We inserted this fragment between the unique Bpu10I and Spe I sites of pCMV-SFV-GCAP to generate pCMV-SFVG-∆1672. The VLV titers obtained after transfection of this DNA onto cells were only 2 × 105 per mL (Fig. 3) indicating that the deletion had only a small effect on increasing VLV titers. We then used reverse transcription and PCR of p50 VLV RNA to generate DNA fragments spanning the indicated restriction sites, EcoRV-BstEII, BstEII-BglII, and BglII-Bpu10I. These fragments containing the p50 VLV mutations were then used to replace the corresponding fragments in the pCMV-SFVG-∆1672 plasmid DNA to generate the recombinants labeled R1, R2, and R2,3. All constructs were sequenced, and we chose only those that matched the p50 VLV consensus. Interestingly, after transfection of these initial recombinants onto cells, they yielded low and variable titers of VLVs (Fig. 3). However, when we put all of the mutations together in recombinant R1,2,3 (plasmid designated pCMV-SFVG-p50R), the high-titer phenotype was recovered. These results indicated that some combination of evolved mutations was required to generate the high-titer phenotype. Also, one construct with a subset of the enhancing mutations (R1 recombinant) grew poorly. We also determined in later constructs that the mutation N34D in the VSV G ectodomain was not required for generation of the high titer VLVs.
Fig. 3.
Reconstruction of the p50 VLV genome shows that multiple mutations are required to generate the high-titer phenotype. The diagram illustrates the pathway of reconstruction of the p50 VLV genome sequence into the pCMV vector used to derive VLVs. The VLV titers obtained at 40 h after transfection of each construct into BHK cells are given on the corresponding line. Range of titers in two experiments is given.
To determine whether the enhancing mutations were affecting replicon RNA synthesis we performed Northern blots to detect replicon RNA or subgenomic G mRNA in cells infected with VLVs derived from pCMV-SFVG-∆1672 or pCMV-SFVG-p50R. We saw no significant differences in the amounts of RNA or in the ratio of replicon to subgenomic RNA in the cells (Fig. S1).
TEM of Infected Cells Indicates a Mechanism for Generation of the High-Titer VLVs.
In our original studies of the VLVs generated by expression of VSV G protein from the SFV replicon, the mechanism that generated the low-titer infectious vesicles was not determined. A possible explanation was that they were derived from SFV replication complexes that had been seen largely in membrane invaginations called spherules within cytopathic vacuoles (CPVs), but also occasionally on the cell surface (10, 18). More recent studies have shown that SFV spherules initially form at the cell surface, and are then endocytosed, eventually accumulating in the cytoplasmic CPVs (9).
To examine the budding of the low vs. high titer VLVs, we prepared VLVs from the construct containing the 3′ deletion (pCMV-SFVG-∆1672) and from the R,1,2,3 construct (pCMV-SFVG-p50R) containing the deletion and the 16 point mutations (Fig. 3). We used these VLVs to infect cells and performed TEM of fixed, thin sections of cells at seven hours after infection. The plasma membranes of cells infected with the low titer (∆1672) VLVs were studded with numerous ∼60- to 70-nm spherical structures that often appeared to be attached to the cell surface by a thin “neck” (Fig. 4 A and B, arrows). We also found occasional intracellular vacuoles that are the typical CPVs containing SFV spherules attached by a neck to the membrane (Fig. 4C). The structures on the cell surface appeared virtually identical to the spherules seen in the CPVs, often including a dense central spot that is thought to be RNA (10).
Fig. 4.
Thin-section TEM of cells infected with VLVs. (A and B) Examples of large numbers of spherule-like structures on the surface of cells infected with low-titer VLVs derived from cells transfected with the pCMV-SFVG-∆1672 plasmid. (C) A typical CPV seen in a cell infected with VLVs. (D and E) Rare examples of what appear to be particles budding from cells infected with high-titer VLVs derived from cells transfected with the pCMV-SFVG-p50R plasmid. Arrows indicate examples of necks apparently attaching spherule-like structures to the membrane.
In contrast to the results with the low titer VLVs, cells infected with the high titer p50 VLVs showed only occasional vesicles that appeared to be budding from the cell surface (Fig. 4 D and E). In many cases these were connected by a neck-like structure (arrows). These results suggested that the p50 VLVs might be budding efficiently, whereas the low-titer VLVs remained mostly attached at the cell surface.
A Late Domain Motif Evolved in the p50 VLV nsP1 Protein.
Many enveloped viruses that bud from the cell surface recruit a cellular budding machinery (ESCRT complex) that cells use to sort cargo into vesicles that bud into multivesicular bodies (12, 13). Viruses that use this pathway have sequence motifs called late domains in their internal structural proteins that recruit components of the ESCRT complex to the budding site. Mutations in these motifs result in incomplete budding of virus particles from the cell and retention by a membranous neck (13). Because the low titer VLVs appeared to retain large numbers of spherule-like structures on the cell surface, we examined the sequence changes in the p50 VLV genome to determine whether the passaging might have selected for a late domain sequence in any of the SFV nonstructural proteins. We found that one of the four mutations in nsP1 changed the amino acid sequence PIAP to PTAP near the C terminus of nsP1 (Table 1). P(T/S)AP is a common motif in internal viral structural proteins that binds to the protein TSG101 to initiate recruitment of an ESCRT protein complex and facilitate virus budding (19).
The PTAP Motif Is Necessary but Not Sufficient for High-titer Particle Release.
The low VLV titers obtained with the R1 recombinant (Fig. 3) suggested that the PTAP motif was not sufficient to generate the high-titer VLVs. However, this interpretation is confounded by the presence of three other mutations in nsP1 and one in nsP2 that might be deleterious in the absence of the other mutations that evolved in the nsP protein complex. To determine whether the PTAP mutation was necessary for high-titer particle production, we mutated just the PTAP motif in the p50 DNA vector (pCMV-SFVG-p50R) back to the original PIAP sequence. We then performed a one-step growth curve using the otherwise isogenic VLVs containing either the PIAP or PTAP sequences to analyze the kinetics of infectious particle release. The results of this experiment are shown in Fig. 5. Both constructs showed similar kinetics of growth reaching plateaus by 24 h. However, p50-PIAP mutant titer was reduced 50- to 100-fold compared with p50-PTAP at the later time points. These results indicate that the presence of the PTAP motif is important for generating the high titer.
Fig. 5.
PTAP motif is required for high-titer VLV production. Cells were infected at an MOI of 10 with high-titer p50VLVs derived from cells transfected with the pCMV-SFVGp50 plasmid or from a mutated form of the plasmid with the PTAP motif in nsP-1 reverted to PIAP. After a 30-min adsorption of VLVs at 37° the cells were washed twice in PBS and the medium was replaced with fresh DMEM containing 5% FBS. Small samples of the medium were then collected immediately (0 time) and at the indicated times thereafter, placed on ice and titered BHK cells using the infectious center assay.
To determine whether the PTAP mutation was sufficient for generating high titer VLVs, we introduced the mutation generating the PTAP motif alone into the pCMV-SFVG-∆1672 plasmid (Fig. 3) and derived VLVs. The VLV titers obtained after transfection of this DNA onto cells were only 1.1 × 105 per mL, indicating that the PTAP motif alone was not sufficient to drive efficient VLV production.
Discussion
We have described evolution of simple, infectious, VLVs that are generated in cells expressing a single structural protein, VSV G, from an RNA replicon (4). The RNA replicon was derived from an SFV expression vector (8) and encodes the nonstructural proteins that form the RNA-dependent RNA replicase, but none of the SFV structural proteins. Cells containing this replicon express VSV G protein on the cell surface and release low titers (∼105 i.u./mL) of infectious vesicles containing VSV G protein and the replicon. Through extensive passaging we have been able to derive VLVs that grow to titers of over 108 i.u./mL Multiple mutations in the SFV nsPs of the evolved VLVs are required for generation of the high titers including a late domain motif that evolved near the C terminus of the SFV nsP1 protein. This motif was necessary, but not sufficient for production of the high titers. SFV nsP1 protein contains an amphipathic membrane anchor sequence for the replicase complex of nsP1-4 and presumably tethers the nsP1-4 complex near or in the base of the spherules in which RNA replication is occurring (20, 21).
Model for Generation of the Low- and High-Titer VLVs.
Fig. 6 shows a model for generation of the high-titer VLVs. SFV replicons not encoding VSV G are initially formed at the plasma membrane in the light-bulb shaped spherules with the replicase proteins near the base of the spherule neck (Fig. 6A). These spherules are then rapidly endocytosed and ultimately accumulate in the classic cytopathic vacuoles (CPVs) seen in cells containing SFV replicons (9). In the case of SFV replicons that encode VSV G, endocytosis of the spherules may be inhibited by VSV G (22, 23), resulting in trapping of spherules on the cell surface (Fig. 6B). These spherules cannot bud efficiently, but are occasionally released from the cell surface to infect neighboring cells. They can also be released by sonication of the cells (4). After extensive passaging of VLVs, multiple mutations were selected including one generating a late domain PTAP motif near the C terminus of the SFV nsP1 protein. The mutations in addition to PTAP are perhaps required to create an appropriate structural context allowing access to binding of the ESCRT protein complex and rapid scission of the spherules from the cell surface (Fig. 6C). The requirement for more than one mutation to generate the high-titer VLV phenotype is consistent with the extensive passaging required to evolve it.
Fig. 6.
Model explaining origin of low-titer and evolved, high-titer VLVs. (A) SFV replicons not expressing VSV G first form in the light-bulb shaped spherules at the cell surface and are then rapidly endocytosed, forming classic CPVs. (B) Expression of VSV G protein from the SFV replicon inhibits endocytosis of the spherules resulting in spherule accumulation at the cell surface. Occasional release of spherules containing VSV G generates low-titer, infectious VLVs that can infect new cells. (C) Extensive passaging of the VLVs selects for mutations that cause rapid VLV release from the cell surface. These high-titer VLVs evolved multiple mutations including a late domain (PTAP) motif in nsP1 that promotes efficient VLV budding and high titers.
Our model is based in part on our observation that large numbers of ∼60- to 70-nm spherule-like structures are present on the surfaces of cells infected with low titer VLVs, whereas cells infected with VLVs containing the PTAP and other mutations showed only occasional vesicles apparently budding from the cell surface. A similar accumulation of HIV-1 and other viruses is seen when viruses have mutations in their late domain motifs (13, 24). If the VLVs were derived exclusively from the budding of spherules, one might expect the purifed p50 VLVs to have a more uniform diameter. However, the purified VLVs that we observed here and previously by TEM (4) ranged in diameter from about 50–200 nm. One possible explanation for the range of sizes is fusion of two or more VLVs, potentially catalyzed by VSV G, to generate larger vesicles. It is also possible that only the smaller VLVs are derived from spherules, and that the larger vesicles are generated through some other mechanism.
All positive-strand RNA viruses use some type of membrane-bound compartments to sequester their RNA during replication. For some viruses there is evidence that these compartments are lined with a shell of replicase proteins (25). In the case of SFV, there is no evidence that the spherules contain a replicase protein shell. The nsPs appear to be located on the cytoplasmic side of the spherule neck (10). The membrane invaginations formed in the generation of multivesicular bodies by the ESCRT pathway lack an internal protein shell (26) and resemble the SFV spherules (27). Thus, it is possible that some components of the ESCRT pathway are normally involved with SFV spherule formation, but the components required for membrane scission are lacking or inhibited. The mutations selected in the p50 VLVs could result in recruitment of additional components required for scission or might release the inhibition of scission. By silver stain (Fig. 2) we did not detect structural proteins other than VSV G in the p50 VLVs suggesting that there is no major component forming a protein shell within them.
It is interesting to speculate that at some early stage in the evolution of enveloped RNA viruses, spherules may have been the direct precursors of primitive virus particles that budded from cells and lacked capsids. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and shielding from recognition by innate immune mechanisms.
Our results led us to search the literature to determine whether there are any known membrane-enveloped RNA viruses that lack a nucleocapsid. Interestingly several positive-strand RNA viruses in the Pegivirus genus of the Flaviviridae family do not appear to encode a capsid protein (28, 29). However, biophysical evidence indicates that they acquire a capsid, perhaps a cellular protein, through an unknown mechanism (30).
In parallel studies we have been examining the immunogenicity and potential pathogenicity of the p50 VLVs. We find that these VLVs retain immunogenicity and lack pathogenicity. Thus, these evolved VLVs have significant potential as vaccine vectors.
Materials and Methods
Reconstruction of the p50 VLV Genome into a Plasmid DNA.
The SuperScript III RT-PCR kit from Life Technologies and 15 DNA primer pairs were used with RNA from p50VLVs to generate overlapping dsDNA fragments covering the p50 VLV genome. Sequences of the fragments were determined (Yale Keck Facility) and assembled using DNAstar software. The plasmid pCMV-SFVG-CAP (Fig. 1) was generated by replacing the SIV gag gene from plasmid pBK-SFVG-E660Gag (6) with a synthetic gene encoding the SFV capsid protein. The 1,672-nucleotide deletion found in the p50 VLV genome sequence was introduced in pCMV-SFVG-CAP (Fig. 3) using a synthetic DNA fragment spanning the Bpu10I-SpeI sites. The remaining mutations were introduced step-wise using the indicated restriction fragments prepared by RT-PCR from p50 VLV RNA. Specific point mutations were generated using the Quik-Change II mutagenesis kit from Agilient Technologies.
Titering of VLVs.
The p50 VLVs were routinely titered using serial dilutions and a standard 2-d plaque assay on BHK-21 (ATCC CCL 10) cell monolayers. Low-titer VLVs plaques were counted using a dissecting microscope or were titered using indirect immunofluorescence microscopy to detect VSV G protein expression in infectious centers.
Purification of p50 VLVs.
Approximately 107 BHK-21 cells in DMEM with 5% (wt/vol) FBS were infected with p50 VLVs at an MOI of 10 and incubated for 24 h at 37°. The medium was then centrifuged at 800 × g for 5 min to remove cells and cell debris. Ten ml of medium was then layered onto 28 mL of 10% (wt/vol) sucrose in PBS and centrifuged at 25,000 rpm for 1.25 h in a Beckman SW28 rotor. The pellet of VLVs was resuspended in 200 μL of PBS and then layered onto 4.5 mL of 10% sucrose in PBS and centrifuged at 40,000 rpm for 1 h in a Beckman SW41 rotor. The pellet was resuspended in 100 μL of PBS. VSV virions were grown and purified in parallel to provide markers for SDS/PAGE gel electrophoresis.
Electron Microscopy of VLVs and VLV-Infected Cells.
VLVs prepared as above (∼5 μL) were applied to carbon-coated EM grids made hydrophobic by plasma ionization. Grids were fixed in 1% glutaraldehyde for 2 min, followed by washing in PBS (pH 7.4) and water, then stained with 2% (wt/vol) uranyl acetate and air-dried. For immunogold labeling, the samples were applied to grids that had been preincubated with PBS containing 1% BSA. Unfixed samples on girds were incubated directly with a mouse monoclonal antibody to VSV G, washed with PBS containing 1% BSA and incubated with donkey anti-mouse IgG conjugated to 12nm gold particles (Abcam). Grids were then washed and stained with 2% (wt/vol) phosphotungstic acid and then dried. Grids were viewed in a FEI 120kV transmission electron microscope. The digital images were acquired with a Gatan 4k × 4k CCD camera.
BHK cells infected with VLVs were fixed at room temperature for 1 h in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2% (wt/vol) glutaraldehyde. After rinsing with the same buffer, cells were postfixed in 0.5% OsO4 at room temperature for 30 min. Specimens were then stained en bloc with 2% aqueous uranyl acetate for 30 min, dehydrated in a graded series of ethanol to 100%, and embedded in Poly/bed 812 for 24 h. Thin sections (60 nm) were cut with a Leica ultramicrotome and poststained with uranyl acetate and lead citrate. Sample grids were examined in a FEI Tencai Biotwin transmission electron microscope at 80 kV. Images were taken using a Morada CCD camera fitted with iTEM (Olympus) software.
Supplementary Material
Acknowledgments
We thank Robert Means and Michael Robek for helpful discussions and suggestions. We thank Gunilla Karlsson, Peter Liljestrom and Margaret Kielian for plasmids and reagents. This work was supported by NIH Grants R37AI-040357 and R01AI-045510 (to J.K.R.)
Footnotes
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1414991111/-/DCSupplemental.
References
- 1.Rossmann MG. Structure of viruses: A short history. Q Rev Biophys. 2013;46(2):133–180. doi: 10.1017/S0033583513000012. [DOI] [PubMed] [Google Scholar]
- 2.Marsh M, Helenius A. Virus entry: Open sesame. Cell. 2006;124(4):729–740. doi: 10.1016/j.cell.2006.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Rose J, Whitt M. Rhabdoviridae: The Viruses and Their Replication. In: Knipe D, Howley P, editors. Fields' Virology. Lippencott-Raven; Philadelphia: 2001. pp. 1221–1240. [Google Scholar]
- 4.Rolls MM, Webster P, Balba NH, Rose JK. Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA. Cell. 1994;79(3):497–506. doi: 10.1016/0092-8674(94)90258-5. [DOI] [PubMed] [Google Scholar]
- 5.Rose NF, Publicover J, Chattopadhyay A, Rose JK. Hybrid alphavirus-rhabdovirus propagating replicon particles are versatile and potent vaccine vectors. Proc Natl Acad Sci USA. 2008;105(15):5839–5843. doi: 10.1073/pnas.0800280105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Schell JB, et al. Significant protection against high-dose simian immunodeficiency virus challenge conferred by a new prime-boost vaccine regimen. J Virol. 2011;85(12):5764–5772. doi: 10.1128/JVI.00342-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Barth BU, Wahlberg JM, Garoff H. The oligomerization reaction of the Semliki Forest virus membrane protein subunits. J Cell Biol. 1995;128(3):283–291. doi: 10.1083/jcb.128.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Liljeström P, Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
- 9.Spuul P, Balistreri G, Kääriäinen L, Ahola T. Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest Virus replication complexes from the plasma membrane to modified lysosomes. J Virol. 2010;84(15):7543–7557. doi: 10.1128/JVI.00477-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Froshauer S, Kartenbeck J, Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol. 1988;107(6 Pt 1):2075–2086. doi: 10.1083/jcb.107.6.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Rossman JS, Lamb RA. Viral membrane scission. Annu Rev Cell Dev Biol. 2013;29:551–569. doi: 10.1146/annurev-cellbio-101011-155838. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Votteler J, Sundquist WI. Virus budding and the ESCRT pathway. Cell Host Microbe. 2013;14(3):232–241. doi: 10.1016/j.chom.2013.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Weiss ER, Göttlinger H. The role of cellular factors in promoting HIV budding. J Mol Biol. 2011;410(4):525–533. doi: 10.1016/j.jmb.2011.04.055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bruce EA, et al. Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway. Virology. 2009;390(2):268–278. doi: 10.1016/j.virol.2009.05.016. [DOI] [PubMed] [Google Scholar]
- 15.Taylor GM, Hanson PI, Kielian M. Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding. J Virol. 2007;81(24):13631–13639. doi: 10.1128/JVI.01688-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Watanabe R, Lamb RA. Influenza virus budding does not require a functional AAA+ ATPase, VPS4. Virus Res. 2010;153(1):58–63. doi: 10.1016/j.virusres.2010.07.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Karlsson GB, Liljeström P. Delivery and expression of heterologous genes in mammalian cells using self-replicating alphavirus vectors. Methods Mol Biol. 2004;246:543–557. doi: 10.1385/1-59259-650-9:543. [DOI] [PubMed] [Google Scholar]
- 18.Grimley PM, Berezesky IK, Friedman RM. Cytoplasmic structures associated with an arbovirus infection: Loci of viral ribonucleic acid synthesis. J Virol. 1968;2(11):1326–1338. doi: 10.1128/jvi.2.11.1326-1338.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Garrus JE, et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell. 2001;107(1):55–65. doi: 10.1016/s0092-8674(01)00506-2. [DOI] [PubMed] [Google Scholar]
- 20.Spuul P, et al. Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication. J Virol. 2007;81(2):872–883. doi: 10.1128/JVI.01785-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Varjak M, Zusinaite E, Merits A. Novel functions of the alphavirus nonstructural protein nsP3 C-terminal region. J Virol. 2010;84(5):2352–2364. doi: 10.1128/JVI.01540-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Whitaker-Dowling P, Youngner JS, Widnell CC, Wilcox DK. Superinfection exclusion by vesicular stomatitis virus. Virology. 1983;131(1):137–143. doi: 10.1016/0042-6822(83)90540-8. [DOI] [PubMed] [Google Scholar]
- 23.Wilcox DK, Whitaker-Dowling PA, Youngner JS, Widnell CC. Rapid inhibition of pinocytosis in baby hamster kidney (BHK-21) cells following infection with vesicular stomatitis virus. J Cell Biol. 1983;97(5 Pt 1):1444–1451. doi: 10.1083/jcb.97.5.1444. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Göttlinger HG, Dorfman T, Sodroski JG, Haseltine WA. Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci USA. 1991;88(8):3195–3199. doi: 10.1073/pnas.88.8.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.den Boon JA, Ahlquist P. Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol. 2010;64:241–256. doi: 10.1146/annurev.micro.112408.134012. [DOI] [PubMed] [Google Scholar]
- 26.Wollert T, Hurley JH. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature. 2010;464(7290):864–869. doi: 10.1038/nature08849. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Kallio K, et al. Template RNA length determines the size of replication complex spherules for Semliki Forest virus. J Virol. 2013;87(16):9125–9134. doi: 10.1128/JVI.00660-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Simons JN, Desai SM, Schultz DE, Lemon SM, Mushahwar IK. Translation initiation in GB viruses A and C: Evidence for internal ribosome entry and implications for genome organization. J Virol. 1996;70(9):6126–6135. doi: 10.1128/jvi.70.9.6126-6135.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Stapleton JT, Foung S, Muerhoff AS, Bukh J, Simmonds P. The GB viruses: A review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae. J Gen Virol. 2011;92(Pt 2):233–246. doi: 10.1099/vir.0.027490-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Xiang J, et al. Characterization of hepatitis G virus (GB-C virus) particles: Evidence for a nucleocapsid and expression of sequences upstream of the E1 protein. J Virol. 1998;72(4):2738–2744. doi: 10.1128/jvi.72.4.2738-2744.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.