Protection of mice against lethal coxsackievirus B3 infection by using DNA immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.     

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

The nucleotide sequences of the genome segments A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3039 nucleotides (1012 deduced amino acids) and 2640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.     

Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

The genetic site(s) that naturally determine the cardiovirulence phenotype of coxsackievirus B3 (CVB3) have yet to be mapped. Using two closely related CVB3 strains that differed in terms of cardiovirulence phenotype in mice, we previously reported the difference in phenotype mapped to a single site, nucleotide 234 (nt234) in the 5' non-translated region (NTR) of the CVB3 genome. When nt234 was C, the virus was attenuated and when U, the virus was cardiovirulent. To determine whether this finding was applicable to other strains of CVB3, we examined 13 different naturally occurring CVB3 strains isolated in different years in the United States. We determined that only two isolates induced severe inflammatory heart muscle disease in C3H/HeJ male mice. Using PCR products as sequencing templates, we determined the 5' NTR sequence from each viral genome. Alignment of these sequences and other published CVB3 5' NTR sequences suggests as many as four separate lineages, with commonly used laboratory strains clustering closely in one branch. An examination of the sequences showed that regardless of cardiovirulence phenotype, nt234 was invariably uridine. Thus, the previously reported cytidine at nt234 is most likely the result of a rare mutation and is not a naturally occurring variation and other sites must account for the variance in virulence seen in natural isolates of CVB3.     

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures

An infectious cDNA of a highly myocarditic coxsackievirus B3 (CVB3m; Nancy strain) was cloned. Sequence data revealed 43 extra non-viral nucleotides upstream of the initial 5' sequence. However, the authentic 5' end sequence was maintained during replication of viral RNA transfected into HeLa cells, suggesting the RNA synthesizing complex edits the picornaviral 5' terminus sequence. Nucleotide sequences of the 5' nontranslated region and the capsid protein gene sequence of CVB3m were compared with the published sequences of five other CVB3 Nancy strains and two main lineages were found. In comparative assays for cardiovirulence, three of four CVB3 tested were cardiovirulent in adolescent male CD-1 mice. Only one of the three available CVB3 strains was neutralized with several anti-CVB3m monoclonal antibodies, suggesting that mutations in the surface epitopes of the capsid polypeptides contribute to antigenic drift within the serotype, perhaps in part through immunoselective pressures. Thus, phenotypic diversity of CVB3 within the prototype Nancy strain is an example of RNA viruses adapting to changing environments (cells, mice and humans) through mutations and selective pressure.     

G (VP7) serotype-dependent preferential VP7 gene selection detected in the genetic background of simian rotavirus SA11

We previously found the preferential selection of VP7 gene from a parent rotavirus strain SA11 with G serotype 3 (G3) in the sequential passages after mixed infection of simian rotavirus SA11 and SA11-human rotavirus single-VP7 gene-substitution reassortants with G1, G2, or G4 specificity. However, it has not been known whether or not VP7 genes derived from other strains with G3 specificity (G3-VP7 gene) are preferentially selected in the genetic background of SA11. To address this question, mixed infections followed by multiple passages were performed with a reassortant SA11-L2/KU-R1 (SKR1) (which possesses VP7 gene derived from G1 human rotavirus KU and other 10 genes of SA11 origin) and one of the five G3-rotaviruses, RRV, K9, YO, AK35, and S3. After the 10th passage, selection rates of SA11-L2/KU-R1 gene 9 (G1-VP7 gene) and gene 5 (NSP1 gene) reduced considerably (0 to 20.4%) in the clones obtained from all the coinfection experiments, while all or some of other segments were preferentially selected from SKR1 depending on the pairs of coinfection. When viral growth kinetics was examined, SKR1 exhibited better growth and reached a higher titer than any G3 viruses. Although the generated reassortants with VP7 gene and NSP1 gene derived from G3 viruses showed almost similar growth kinetics to that of SKR1 during the first 20 h of replication, the titers of these reassortants were higher than that of SKR1 after 36 h postinfection. The results obtained in this study suggested that G3-VP7 gene is functionally more adapted to the genetic background of SA11.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.     

Coxsackieviral proteins functionally recognize the polioviral cloverleaf structure of the 5'-NTR of a chimeric enterovirus RNA: influence of species-specific host cell factors on virus growth

The 5'-non-translated region (NTR) of enteroviruses contains secondary structures which do not only serve in the initiation of translation but also in the initiation of plus-strand RNA synthesis by binding of viral and cellular proteins. To investigate a very early step of enteroviral replication by cis- and trans-complementation, 220 nucleotides of the 5'-region of coxsackievirus B3 (CVB3) were exchanged with the corresponding region of poliovirus type 1 (PV1) to yield the chimeric virus CVB3[PV5']. The viability of this chimera demonstrates that the polioviral cloverleaf structure of the 5'-NTR is functional in the replication of a chimeric CVB3 RNA. The HeLa-generated chimera reveals a 4-nucleotide deletion (nt 232-235) within a short direct repeat. Besides clearly reduced growth characteristics in all permissive cell lines, the chimera exhibits a small-plaque phenotype. The host range is changed since the virus grows well in human HeLa cells, but does not replicate in murine YAC-1 and Ltk cells, although these cell lines are permissive for the replication of both parental viruses. Moreover, in simian Vero, COS-1, or FRhK-4 cells the HeLa-generated chimera CVB3[PV5'] exhibits a strict temperature sensitivity at 39 degrees C. After infection of simian cells with high m.o.i. in situ hybridization data reveal that the chimera replicates in single cells at almost normal rates indicating that only a small fraction of HeLa-generated virus is able to multiplicate in simian cell lines. After passaging the virus chimera in Vero cells two further mutations occur at nucleotide positions 185 and 227. Since this genome region is known to interact with viral proteins and several host cell factors during the initiation of replication and translation, interactions of such factors with either viral RNA or viral proteins may be disturbed but still functional at permissive temperatures in HeLa cells and simian cell lines, whereas murine cell lines are not permissive. These experiments suggest that phenomena like host range, tissue tropism and cell-type specificity may be explained as a complex interplay of cellular surface receptors and intracellular host factors. Such intracellular factors could be part of the enteroviral initiation complex during the plus-strand RNA synthesis or during translation initiation and could be expressed in a tissue-, organ- or species-specific way or might be regulated developmentally.     

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.     

Sequences responsible for the distinctive hemolytic potentials of Friend and Moloney murine leukemia viruses are dispersed but confined to the psi-gag-PR region

Friend and Moloney murine leukemia viruses (F- and M-MuLV) induce distinct diseases in hematopoietic tissues following inoculation of newborn mice of susceptible strains. F-MuLV induces erythroleukemia preceded by severe early hemolytic anemia; M-MuLV induces thymomas and only very mild hemolysis. The major viral determinant of severe early hemolytic anemia residues in the env gene, but sequences located outside this gene can modulate this effect. By means of genetic chimeras of F- and M-MuLV, we have found that although they are confined to the 5' portion of the env gene intron, sequences that determine the distinctive hemolytic potentials of F- and M-MuLV are widely distributed over a region spanning the RNA encapsidation domain, the gag gene, and the portion of the pol gene encoding the viral protease. Within this large region, two fragments of M-MuLV, a 1.3-kb region encoding the matrix, pp12, and capsid proteins and a 0.8-kb region encoding the nucleocapsid and the viral protease, were capable, individually, of partially attenuating the capacity of F-MuLV for induction of severe early hemolytic anemia. In association, these two fragments conferred complete attenuation. Moreover, a second pair of adjacent fragments within this large region appeared to behave cooperatively to confer complete attenuation; a 0.36-kb region roughly corresponding to the encapsidation domain, although not detectably altering hemolytic potential on its own, deepened the attenuation conferred by the adjacent 1.3-kb region. Whether capable of inducing severe early hemolytic anemia or not and despite different efficiencies of induction of recombinant polytropic viruses, all chimeric viruses retained the erythroleukemogenicity of the F-MuLV parent.     

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.     

Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.     

Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.     

Capsid protein VP1 deletions in JC virus from two AIDS patients with progressive multifocal leukoencephalopathy

PCR on 52 cerebrospinal fluid (CSF) specimens and 33 brain biopsies obtained from HIV-1 positive patients utilized pairs of primers from both the early region (JTP) and late region (JLP). In these patients, in whom progressive multifocal leukoencephalopathy (PML) was suspected on the basis of clinical symptoms and magnetic resonance imaging (MRI) studies, eight CSFs (15%) and 14 brain biopsy specimens (42%) contained JCV DNA sequences. In two patients' samples, the CSFs were positive for JCV DNA in the VP1 region using the primer pair for the VP1 region (JLP), but the fragment amplified migrated more rapidly than the 129-bp product obtained from prototype JCV(Mad-1) or the fragment amplified from the antigenic variant of JCV known as Mad-11. These patients died 3-4 months after onset of progressive neurological symptoms. Cycle sequencing of the fragments revealed overlapping deletions of 24 and 27 nucleotides. These strains were of different genotypes, designated strain 107 and strain 206. Computer analysis of the VP1 amino acid sequence predicts that the eight or nine amino acid residue deletions represent a surface loop with a high antigenic index. These naturally occurring deletion mutants are the first examples of a phenomenon observed experimentally in the mouse polyoma virus capsid protein VP2.     

Attachment and growth of human rotaviruses RV-3 and S12/85 in Caco-2 cells depend on VP4

Studies with human neonatal rotaviruses RV-3 and S12/85 and their reassortants showed that VP4 is a determinant of rotavirus attachment to and growth in Caco-2 cells. The binding of these viruses to MA104 and Caco-2 cells correlated with their growth ability. Virus sensitivity to trypsin and the VP4 fusion region may be implicated in these processes.