Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C

We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4(+)-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.     

Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.     

The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies

Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and S2. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the S2 gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the S2 gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344-8348, 1998). We now describe in vivo studies that examine the evolution and role of the S2 gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that the S2 gene is highly conserved during persistent infection and that deletion of the S2 gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional S2 gene. These data indicate that the EIAV S2 gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of S2 influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAV S2 that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.     

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.     

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.     

Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant

Translation initiation factors are universal determinants of plant susceptibility to RNA viruses, but the underlying mechanisms are poorly understood. Here, we show that a sequence in the 3' untranslated region (3'-UTR) of a viral genome that is responsible for overcoming plant eIF4E-mediated resistance (virulence determinant) functions as a 3' cap-independent translational enhancer (3'-CITE). The virus/plant pair studied here is Melon necrotic spot virus (MNSV) and melon, for which a recessive resistance controlled by melon eIF4E was previously described. Chimeric viruses between virulent and avirulent isolates enabled us to map the virulence and avirulence determinants to 49 and 26 nucleotides, respectively. The translational efficiency of a luc reporter gene flanked by 5'- and 3'-UTRs from virulent, avirulent and chimeric viruses was analysed in vitro, in wheatgerm extract, and in vivo, in melon protoplasts, showing that: (i) the virulence determinant mediates the efficient cap-independent translation in vitro and in vivo; (ii) the avirulence determinant was able to promote efficient cap-independent translation in vitro, but only when eIF4E from susceptible melon was added in trans, and, coherently, only in protoplasts of susceptible melon, but not in the protoplasts of resistant melon; (iii) these activities required the 5'-UTR of MNSV in cis. Thus, the virulence and avirulence determinants function as 3'-CITEs. The activity of these 3'-CITEs was host specific, suggesting that an inefficient interaction between the viral 3'-CITE of the avirulent isolate and eIF4E of resistant melon impedes the correct formation of the translation initiation complex at the viral RNA ends, thereby leading to resistance.     

Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication

The 5' portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5' 200 bases of the SIN genome RNA, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5' end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5' terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5' end of the original SFV 5' sequence or its "deleted" versions. Early pseudorevertants had heterogeneous 5' termini that were inefficient for replication relative to the parental SIN 5' sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5'-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5'-terminal sequences always began with 5'-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5' modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.     

Biologic Studies of Chimeras of Highly and Moderately Virulent Molecular Clones of Simian Immunodeficiency Virus SIVsmPBj Suggest a Critical Role for Envelope in Acute AIDS Virus Pathogenesis

Previous studies identified three molecular clones of the acutely pathogenic SIVsmPBj strain that varied in terms of relative in vivo pathogenicity. One clone, SIVsmPBj6.6, reproducibly induced a rapidly fatal disease in pigtailed macaques. In contrast, a highly related clone (SIVsmPBj6.9) was only minimally pathogenic in macaques. PBj6.6 and PBj6.9 shared a tyrosine substitution at position 17 in the Nef protein that is a major determinant of virulence but differed at one residue in Vpx (C89R), three residues within the envelope (D119G, R871G, G872R), and a single residue in Nef (F252L). SIVsmPBj6.9 was less efficient in inducing proliferation of resting macaque peripheral blood mononuclear cells in vitro than SIVsmPBj6.6 and exhibited a marked reduction in infectivity relative to SIVsmPBj6.6. Chimeric viruses for each of these variable residues were constructed, and their biologic properties were compared to those of the parental strains. Differences in Vpx and Nef did not alter the basic biologic phenotype of the chimeras. However, the D119G substitution in the envelope of SIVsmPBj6.9 was associated with a marked reduction in the infectivity of this virus relative to SIVsmPBj6.6. An associated processing defect in gp160 of SIVsmPBj6.9 and chimeras expressing the D119G substitution suggests that a reduction in virion envelope incorporation is the mechanistic basis for reduced virion infectivity. In vivo studies revealed that substitution of the PBj6.9 amino acid into PBj6.6 (D119) abrogated the pathogenicity of this previously pathogenic virus. Introduction of the PBj6.9 G119, however, did not confer full virulence to the parental PBj6.9 virus, implicating one or all of the other four substitutions in the virulence of SIVsmPBj6.6.     

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.     

Coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization

Despite their high frequency of RNA recombination, the plus-strand coronaviruses have a characteristic, strictly conserved genome organization with the essential genes occurring in the order 5'-polymerase (pol)-S-E-M-N-3'. We have investigated the significance of this remarkable conservation by rearrangement of the murine coronavirus genome through targeted recombination. Thus, viruses were prepared with the following gene order: 5'-pol-S-M-E-N-3', 5'-pol-S-N-E-M-3', 5'-pol-M-S-E-N-3', and 5'-pol-E-M-S-N-3'. All of these viruses were surprisingly viable, and most viruses replicated in cell culture with growth characteristics similar to those of the parental virus. The recombinant virus with the gene order 5'-pol-E-M-S-N-3' was also tested for the ability to replicate in the natural host, the mouse. The results indicate that the canonical coronavirus genome organization is not essential for replication in vitro and in vivo. Deliberate rearrangement of the viral genes may be useful in the generation of attenuated coronaviruses, which due to their reduced risk of generating viable viruses by recombination with circulating field viruses, would make safer vaccines.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

Characterization of a replication-defective human immunodeficiency virus type 1 att site mutant that is blocked after the 3' processing step of retroviral integration

Two activities of retroviral integrase, 3' processing and DNA strand transfer, are required to integrate viral cDNA into a host cell chromosome. Integrase activity has been analyzed in vitro using purified protein and recombinant DNA substrates that model the U3 and U5 ends of viral cDNA or by using viral preintegration complexes (PICs) that form during virus infection. Numerous studies have investigated changes in integrase or viral DNA for effects on both 3' processing and DNA strand transfer activities using purified protein, but similar analyses have not been carried out using PICs. Here, we analyzed PICs from human immunodeficiency virus type 1 (HIV-1) strain 604del, an integration-defective mutant lacking 26 bp of U5, and revE1, a revertant of 604del containing an additional 19-bp deletion, for levels of 3' processing activity that occurred in infected cells and for levels of in vitro DNA strand transfer activity. Whereas revE1 supported one-third to one-half of the level of wild-type DNA strand transfer activity, the level of 604del DNA strand transfer activity was undetectable. Surprisingly, integrase similarly processed the 3' ends of 604del and revE1 in vivo. We therefore conclude that 604del is blocked in its ability to replicate in cells after the 3' processing step of retroviral integration. Whereas Western blotting showed that wild-type, revE1, and 604del PICs contained similar levels of integrase protein, Mu-mediated PCR footprinting revealed only minimal protein-DNA complex formation at the ends of 604del cDNA. We propose that 604del is replication defective because proteins important for DNA strand transfer activity do not stably associate with this cDNA after in vivo 3' processing by integrase.     

Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIVSF33A Molecular Clones

In vivo adaptation of simian-human immunodeficiency virus (SHIV) clone SHIV(SF33) resulted in the emergence of pathogenic isolate SHIV(SF33A), which caused a rapid and severe CD4(+) T-cell depletion when inoculated into rhesus macaques. Two molecular clones generated by inserting the env V1-to-V5 region amplified from SHIV(SF33A)-infected animals into the parental SHIV(SF33) genome retained a pathogenic phenotype. The gp120 envelope glycoproteins of pathogenic clones SHIV(SF33A2) and SHIV(SF33A5) conferred a threefold increase in viral entry and fusogenicity compared to the parental glycoprotein. Changes in gp120 were also responsible for a higher replication capacity and cytopathicity in primary CD4(+) T-cell cultures. Last, gp120 carried the determinants of SHIV(SF33A) neutralization resistance. Thus, changes in SHIV(SF33A) gp120 produced a set of properties that could account for the pathogenic phenotype observed in vivo. Measurement of antibody binding to SHIV(SF33A) viral particles revealed an increased exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody in a region that was shown to contribute to coreceptor binding. Exposure of this epitope occurred in the absence of CD4 binding, suggesting that the envelope glycoprotein of pathogenic SHIV(SF33A) clones folded in a conformation that was primed for interaction with CXCR4 or for the subsequent step of fusion.     

Stereospecificity of reactions catalyzed by HIV-1 integrase

The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.