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PubMed2000

A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA.

Bovine viral diarrhea virus (BVDV), a Pestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5' and 3' ends. The 5' UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5'-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5' end of the viral genome (hairpin Ia) as well as at the 3' end of the complementary negative-strand replication intermediate [termed hairpin Ia (-)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5'-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (-) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (-) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5'-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.

5' Untranslated Regions↗
PubMed2000

Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates.

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955-2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503-510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

Amino Acid Sequence↗
PubMed2000

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype.

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Amino Acid Sequence↗
PubMed2000

Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene.

Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.

Base Sequence↗
PubMed2000

Identification of a minimal size requirement for termination of vesicular stomatitis virus mRNA: implications for the mechanism of transcription.

The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3'-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5' modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.

Base Sequence↗
PubMed2000

Sendai virus C proteins must interact directly with cellular components to interfere with interferon action.

Sendai virus (SeV) infection of interferon (IFN)-competent cells is one of the most efficient ways of inducing IFN production. Virus replication is nevertheless largely unaffected, since SeV infection also interfers with IFN action, a prerequisite for the establishment of an antiviral state. This property has been mapped by reverse genetics to the viral C gene, which is also known to act as a promoter-specific inhibitor of viral RNA synthesis. Using luciferase reporter plasmids containing IFN-responsive promoters, we have found that all four C proteins effectively interdict IFN signaling when expressed independently of SeV infection. The C proteins must therefore interact directly with cellular components to carry this out. The C gene in the context of an SeV infection was also found to induce STAT1 instability in some cells, whereas in other cells it apparently acts to prevent the synthesis of STAT1 in response to the virus infection or IFN treatment. The SeV C proteins appear to act in at least two ways to counteract the IFN induced by SeV infection.

Animals↗
PubMed2000

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure.

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

Amino Acid Sequence↗
PubMed2000

In vitro- and in vivo-generated defective RNAs of satellite panicum mosaic virus define cis-acting RNA elements required for replication and movement.

Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

3' Untranslated Regions↗
PubMed2000

NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs.

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.

Animals↗
PubMed2001

A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos.

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

Animals↗
PubMed2001

The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity.

Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV(5804)), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDV(OS) and CDV(OL), respectively), and the MV vaccine strain Edmonston B (MV(Edm)). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDV(OS). The coding regions of the H proteins of all CDV strains and MV(Edm) were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H(5804), rCDV-H(OL), rCDV-H(Edm), rMV-H(5804), rMV-H(OL), and rMV-H(OS) were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family Paramyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.

Animals↗
PubMed2001

A predicted secondary structural domain within the internal ribosome entry site of echovirus 12 mediates a cell-type-specific block to viral replication.

The enterovirus 5' nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5' (m(7)GpppN) cap-independent manner, and cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5' NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5'NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37 degrees C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5'NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5' NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.

5' Untranslated Regions↗
PubMed2001

Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Amino Acid Sequence↗
PubMed2001

RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome.

Arenaviruses have a bisegmented negative-strand RNA genome whose proteomic capability is limited to only four polypeptides, namely, nucleoprotein (NP), surface glycoprotein (GP) that is proteolytically processed into GP1+GP2, polymerase (L), and a small (11-kDa) RING finger protein (Z). The role of Z during the Lymphocytic choriomeningitis virus (LCMV) life cycle is poorly understood. We investigated the function of Z in virus transcription and replication by using a reverse genetic system for the prototypic arenavirus LCMV. This system involves an LCMV minigenome and the minimal viral trans-acting factors (NP and L), expressed from separated cotransfected plasmids. Cotransfection of the Z cDNA strongly inhibited LCMV minigenome expression. The effect required synthesis of Z protein; its magnitude was dose dependent and occurred with levels of Z protein substantially lower than those observed in LCMV-infected cells. Coexpression of Z did not prevent the encapsidation of plasmid supplied minigenome, but it affected both transcription and RNA replication similarly. Mutations in Z that unfolded its RING finger domain eliminated its inhibitory activity, but RING proteins not related to Z did not affect LCMV minigenome expression. Consistent with the minigenome results, cells transiently expressing Z exhibited decreased susceptibility to infection with LCMV.

Animals↗
PubMed2001

Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence.

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E(rns) (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553-9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser(476) and an HS-dependent recombinant virus (S-RT virus) with Arg(476) were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E(rns), E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg(476) was mutated back to Ser(476). Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E(rns) S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.

Animals↗
PubMed2001

Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains.

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template.

Amino Acid Sequence↗
PubMed2001

Recombinant Newcastle disease virus as a vaccine vector.

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.

Animals↗