Recombinant feline herpesviruses expressing feline leukemia virus envelope and gag proteins.

We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp70 and p15E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp70 proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.     

An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.     

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.     

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.     

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

A neutralizing antibody-inducing peptide of the V3 domain of feline immunodeficiency virus envelope glycoprotein does not induce protective immunity.

Specific-pathogen-free cats, immunized with a 22-amino-acid synthetic peptide designated V3.3 and derived from the third variable region of the envelope glycoprotein of the Petaluma isolate of feline immunodeficiency virus (FIV), developed high antibody titers to the V3.3 peptide and to purified virus, as assayed by enzyme-linked immunoassays, as well as neutralizing antibodies, as assayed by the inhibition of syncytium formation in Crandell feline kidney cells. V3.3-immunized animals and control cats were challenged with FIV and then monitored for 12 months; V3.3 immunization failed to prevent FIV infection, as shown by virus isolation, anti-whole virus and anti-p24 immunoglobulin G antibody responses, and positive PCRs for gag and env gene fragments. Sequence analysis of the V3 region showed no evidence for the emergence of escape mutants that might have contributed to the lack of protection. The sera of the V3.3-hyperimmunized cats and two anti-V3.3 monoclonal antibodies neutralized FIV infectivity for Crandell feline kidney cells at high antibody dilutions but paradoxically failed to completely neutralize FIV infectivity at low dilutions. Moreover, following FIV challenge, V3.3-immunized animals developed a faster and higher antiviral antibody response than control cats. This was probably due to enhanced virus replication, as also suggested by quantitative PCR data.     

Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus.

The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses.     

Nucleotide sequences of the envelope genes of two isolates of feline leukemia virus subgroup B

We determined the nucleotide sequences of the envelope genes of the Snyder-Theilen and Gardner-Arnstein isolates of feline leukemia virus subgroup B. Comparison of the deduced amino acid sequences of the envelope gene products revealed regions of sequence divergence, which we relate to structural features of the viral protein. We also examined nucleotide sequences within the long terminal repeats of these related isolates of feline leukemia virus subgroup B.     

Characterization and significance of delayed processing of the feline leukemia virus FeLV-FAIDS envelope glycoprotein.

FeLV-FAIDS, an immunodeficiency-inducing isolate of feline leukemia virus, is composed of a pathogenic but replication-defective genome (molecular clone 61C) and a replication-competent but non-immunodeficiency-inducing variant genome (molecular clone 61E). The chimeric virus EECC, composed of the 5' gag-pol of 61E fused to the env-3' LTR of 61C, also induces immunodeficiency. The 61C (or EECC) gp80 can be distinguished from that of 61E on the basis of antigenic recognition, size, and rate of posttranslational processing. We found that the nascent precursor polypeptides of the two viruses were the same size; however, the 61E gp80 rapidly shifted to a smaller size and was subsequently cleaved to gp70, whereas EECC gp80 maintained its nascent size and was cleaved to gp70 only after a prolonged time. Endo-beta-N-acetyl glucosaminidase H and N-glycanase digestions of newly formed glycoproteins resulted in a similar banding pattern for both viruses, indicating that both contained the same number of oligosaccharide side chains and that all of these were high mannose sugars. The metabolic inhibitors of glycosylation, castanospermine or N-methyldeoxynojirimycin, prevented both the rapid trimming of 61E gp80 and its cleavage to gp70. Treatment with mannosidase inhibitors, however, did not affect 61E gp80 processing or size, suggesting that retention of glucose residues on EECC was responsible for these distinguishing properties of the glycoprotein. The pathological consequence of aberrant viral glycoprotein processing was evaluated in feline 3201 T lymphocytes, which are infectable by both 61E and EECC but are killed only by EECC. As in fibroblasts, the EECC glycoprotein produced in lymphocytes was larger, antigenically distinct, and processed more slowly than was the glycoprotein of 61E. Castanospermine treatment of 61E-infected 3201 T cells, however, not only abrogated the antigenic differences between the 61E and EECC glycoproteins but also resulted in a cytopathic effect. Our results suggest that (i) intracellular accumulation of EECC envelope glycoprotein may occur consequent to retention of glucose residues on carbohydrate side chains and (ii) a strong correlation exists between delayed glycoprotein processing and cytopathicity in FeLV-FAIDS-infected T lymphocytes.     

Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins.

Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

X-ray diffraction study of feline leukemia virus fusion peptide and lipid polymorphism

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using a temperature ramp with sequential X-ray diffraction. This peptide, the hydrophobic amino-terminus of p15E, has been proven to be fusogenic and to promote the formation of highly curved, intermediate structures on the lamellar liquid-crystal to inverse hexagonal phase transition pathway. The FeLV peptide produces marked effects on the thermotropic mesomorphic behaviour of MeDOPE, a phospholipid with an intermediate spontaneous radius of curvature. The peptide is shown to reduce the lamellar repeat distance of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism.     

Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors.     

Analysis of intracellular feline leukemia virus proteins. I. Identification of a 60,000-dalton precursor of feline leukemia virus p30.

The synthesis and release of feline leukemia virus p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with [35S]methionine contained equal amounts of p30 in both the CE and the PF. p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cell pulse labeled for 2.5 min contained a labeled 60,000-dalton polypeptide (Pp60) in the PF and a polypeptide in the CE that comigrated with feline leukemia virus p30 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When cells were chased after a pulse label, there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in both the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5- to 24-h chase period suggested that most of the newly synthesized p30 was incorporated into extracellular virus. Typtic peptide analysis of labeled Pp60 and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp60 molecule. The tryptic peptide analysis in concert with the pulse-chase labeling data provides strong evidence that Pp60 is a precursor of p30.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Topography of murine leukemia virus envelope proteins: characterization of transmembrane components

Trypsinization of intact Moloney murine leukemia virus resulted in cleavage of p15(E) and Pr15(E) at a site near the middle of the molecule, producing a 9,000-dalton amino-terminal fragment which contains the disulfide linkage site to gp70 and which carries p15(E) epitopes b and c, but not epitope a. After solubilization of the viral membrane, trypsinization occurred at a second site within 1,000 daltons of the carboxy end of p15(E). This site is not exposed in intact virions, indicating that p15(E) and Pr15(E) are transmembrane proteins.     

Comparisons of the peptide maps of Kunjin virus proteins smaller than the envelope protein.

We analyzed the maps of [35S]methionine-labeled tryptic peptides of the Kunjin virus-specified proteins NV2 1/2, NV2, V2, NV1 1/2, NV1, and V1. The peptides of NV1 1/2 are identical to those of V2, except for one peptide contained only in the latter. The maps of each of the other proteins are unique and, consequently there is no evidence of any one protein being derived from another by proteolytic cleavage. The tryptic peptide maps of the above polypeptides were also compared with those of the larger Kunjin proteins, NV5, NV4, and V3. The resolution of the peptides of NV2 1/2 and NV1 is adequate to exclude any relationships with NV5 and NV4, but a possible relationship with V3 remains, although it seems unlikely in the light of other evidence. Since the peptide map of V1 is comprised of only two methionine-containing peptides similar in mobilities to two peptides found in the maps of NV5, NV4, and V3, a precursor, if any, of V1 has not been positively identified. The peptides of NV2 and V2 (NV1 1/2) are not contained in digests of NV5, NV4, or V3, and therefore, like the latter, NV2 and V2 (NV1 1/2) are independent products of translation from the positive-strand genome.