Pathogenicity and rapid growth kinetics of feline immunodeficiency virus are linked to 3' elements

Chimeric viruses constructed between a highly pathogenic Feline Immunodeficiency Virus isolate (FIV-C36) and a less pathogenic but neurotropic strain (FIV-PPR) have been used to map viral genetic determinants of in vivo pathogenicity. Chimeric virus FIV-PCenv, which contains FIV-C36 genome from the 3' region of pol to upstream of the 3'LTR on an FIV-PPR backbone, was previously shown to be replication-competent in vivo, inducing altered CD4(+) T-cell and neutrophil profiles intermediate between parental strains following a delay in viral replication during initial infection. Examination of FIV-PCenv proviral sequences recovered at week 11 post-infection revealed two changes compared to initial viral inoculum; the most significant being arginine to histidine in the integrase region of Pol at residue 813 (R813H). Pooled plasma from the initial in vivo study was used to inoculate a second cohort of cats to determine whether similar virulence and kinetics could be established following primary infection. Viral replication kinetics and immunocyte profiles were monitored in blood, bone marrow, and saliva over a one-year period. Passaged FIV-PCenv again displayed intermediate phenotype between parental strains, but unlike primary experiments, the onset of acute viremia was not delayed. CD4/8 alterations were noted in all groups of animals, though significant changes from controls were delayed in FIV-PPR infected animals compared to FIV-C36 and FIV-PCenv. In vivo passage of FIV-PCenv increased replication-competence relative to the initial molecularly-cloned chimera in association with one adaptive nucleotide change in the 5' end of the genome relative to primary tissue culture inoculum, while mutations in the 3' end of the genome were not detected. The results are consistent with the interpretation that 3' elements contribute to heightened virulence of FIV-C36, and that integrase residue 813 plays an important role in facilitating successful in vivo replication.     

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.     

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.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

马传染性贫血病毒基因非编码区LTR嵌合克隆的构建

在已有全长感染性克隆pLGFD3 8 和pD70344 的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换。将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT PCR方法及Real time RT PCR验证其感染性。结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT PCR阳性。电镜下可见大量典型的EIAV颗粒。pLGFD9 12 嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3 8具有相似的复制水平,pLGFD9 12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。     

Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence.

A major determinant of neurovirulence for the GDVII strain of Theiler's virus, a murine picornavirus, was mapped to the P1 capsid protein region. Chimeric viruses were constructed by using sequences from the 5' noncoding and P1 regions of the virulent GDVII strain to replace equivalent regions of the less virulent BeAn strain. Neurovirulence in mice progressively increased as larger regions of BeAn capsid protein-encoding sequences were replaced. The in vitro growth characteristics of the chimeras showed that some chimeras were growth delayed in BHK-21 cells even though the viral constructs exhibited larger plaque sizes, were less temperature sensitive, and were more thermally stable than BeAn. Examination of assembly intermediates revealed an altered pentamer conformation and delayed empty capsid formation for the growth-compromised viruses. For these constructs, their chimeric nature inadvertently resulted in virion assembly defects that complicated finer-scale mapping of the determinants of virulence within the capsid region. These results demonstrate the importance of determining in vitro growth characteristics of chimeras to correctly decipher the significance of their phenotypes. VP1 does not contain a complete determinate for virulence because a chimera with VP1-encoding sequences from GDVII in an otherwise BeAn virus has an attenuated phenotype but is not growth compromised in vitro. The source of sequences, BeAn or GDVII, in the 5' noncoding region had only slight effects on the virulence of recombinant constructs.     

Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.     

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.     

The R-U5-5'' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.     

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.     

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.     

Large RNase T1-resistant oligonucleotides encoding p15E and the U3 region of the long terminal repeat distinguish two biological classes of mink cell focus-forming type C viruses of inbred mice

We used T1 oligonucleotide maps, in conjunction with available nucleotide sequences of appropriate C-type viruses, to identify regions of the viral genome that distinguish two biological classes of mink cell focus-forming (MCF) viruses described previously by Cloyd et al. (J. Exp. Med. 151:542-522, 1980). We found that leukemogenic MCF viruses from thymus differed from non-leukemogenic MCFs isolated from nonthymic neoplasms in nucleotide sequences encoding Prp15E and the U3 portion of the long terminal repeat (LTR). The thymic isolates possessed recombinant Prp15E genes, with the 5' to mid portion derived from their ecotropic parents and the extreme 3' portion invariably derived from their nonecotropic parents. These viruses probably derived the entire U3 portion of their LTRs from their nonecotropic parents. The nonthymic MCFs appeared to inherit their entire Prp15E coding region from their nonecotropic parents. We failed to detect consistent differences in gp70-coding sequences between the two groups of MCFs, but this may simply reflect limitations of the data. The studies presented here, in conjunction with studies from a number of labs indicating a role for MCF gp70 in leukemogenesis, indicate that three genetic elements, gp70, p15E, and the U3 portion of the LTR, may all play a role in determining the leukemogenic phenotype of type C viruses of high-leukemic inbred mice.     

Envelope variants from women recently infected with clade A human immunodeficiency virus type 1 confer distinct phenotypes that are discerned by competition and neutralization experiments

Women infected with clade A human immunodeficiency virus type 1 harbor a virus population that is genetically diverse in the envelope gene, a fact that contrasts with the homogeneous virus population identified in newly infected men. It is not known whether viral genetic diversity at this early stage of infection is manifested as phenotypic diversity. This is a significant question because phenotypic diversity in the viral population that establishes infection in women may have important implications for pathogenesis and therapeutic intervention. Thus, in this study we compared the biological properties of three pairs of chimeric viruses that contained envelope genes representative of variant groups in each of three infected women-Q23, Q45, and Q47. Envelope chimeras were evaluated for replication in stimulated and resting peripheral blood mononuclear cells alone and in competition, for coreceptor use, and for neutralization sensitivity. All viruses utilized CCR5 exclusively and had a non-syncytium-inducing phenotype on MT-2 cells and in primary culture. There were no significant differences in replication parameters between paired variants in individual cultures. However, in competition experiments, one chimera of each variant pair always dominated. The dominant virus from Q23 and Q47, but not from Q45, infected a significantly higher number of CCR5- and CD4-expressing GHOST cells than the weaker chimeras. Significantly, chimeric viruses from Q47 and Q45 showed markedly different neutralization sensitivity to antibodies to CCR5 and gp120, respectively. These data indicate that distinct envelope genotypes identified in clade A-infected women near seroconversion confer unique phenotypes that affect viral fitness and that may be due, in part, to different requirements for relative configuration of CD4 and CCR5 on infected cells.     

Marker rescue of adeno-associated virus (AAV) capsid mutants: a novel approach for chimeric AAV production

Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact rep gene, and a mutated cap gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus dl309 helper virus. However, the mutation in the cap gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The cap genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3 cap gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric cap genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.     

Nsp9 and Nsp10 Contribute to the Fatal Virulence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China

Atypical porcine reproductive and respiratory syndrome (PRRS), which is caused by the Chinese highly pathogenic PRRS virus (HP-PRRSV), has resulted in large economic loss to the swine industry since its outbreak in 2006. However, to date, the region(s) within the viral genome that are related to the fatal virulence of HP-PRRSV remain unknown. In the present study, we generated a series of full-length infectious cDNA clones with swapped coding regions between the highly pathogenic RvJXwn and low pathogenic RvHB-1/3.9. Next, the in vitro and in vivo replication and pathogenicity for piglets of the rescued chimeric viruses were systematically analyzed and compared with their backbone viruses. First, we swapped the regions including the 5′UTR+ORF1a, ORF1b, and structural proteins (SPs)-coding region between the two viruses and demonstrated that the nonstructural protein-coding region, ORF1b, is directly related to the fatal virulence and increased replication efficiency of HP-PRRSV both in vitro and in vivo. Furthermore, we substituted the nonstructural protein (Nsp) 9-, Nsp10-, Nsp11- and Nsp12-coding regions separately; or Nsp9- and Nsp10-coding regions together; or Nsp9-, Nsp10- and Nsp11-coding regions simultaneously between the two viruses. Our results indicated that the HP-PRRSV Nsp9- and Nsp10-coding regions together are closely related to the replication efficiency in vitro and in vivo and are related to the increased pathogenicity and fatal virulence for piglets. Our findings suggest that Nsp9 and Nsp10 together contribute to the fatal virulence of HP-PRRSV emerging in China, helping to elucidate the pathogenesis of this virus.     

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6-kb ClaI-KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3' terminus of the env gene, the intergenic region, the U3, and the 5' half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8-LTR contributes to the manifestation of thymoma with rapid progression in rats.     

Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia.

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.     

Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression

A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+) memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 alpha-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.     

Requirements for RNA Replication of a Poliovirus Replicon by Coxsackievirus B3 RNA Polymerase

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D(pol)) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D(pol) from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5' end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33 degrees C, but not at 37 degrees C. Replacement of the PV1 5'-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.