Gail W. Wertz

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome

The RNA-dependent RNA polymerase of the nonsegmented negative-strand RNA viruses carries out two distinct RNA synthetic processes: transcription of monocistronic, capped, and polyadenylated subgenomic messenger RNAs, and replication by means of the synthesis of a full-length positive-sense copy of the genome. The template for both processes is the negative-sense genomic RNA tightly encapsidated by the viral nucleocapsid protein. By applying UV transcriptional mapping to engineered variants of vesicular stomatitis virus, we discovered that, in infected cells, transcription and replication are controlled by initiation at different positions on the viral genome.

The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles

Abstract Infectious defective interfering (DI) particles of the negative-stranded RNA virus vesicular stomatitis virus (VSV) havebeen recovered from negative-sense transcripts of a plasmid that contains a full-length cDNA derived from the DI-T particle genome. In order to determine the cis-acting sequences necessary for RNA replication, encapsidation, and budding and to approximate the minimal size of RNA that can be packaged into infectious particles, we constructed a series of internal deletions in the DI cDNA to generate plasmids that could be transcribed to yield RNAs which ranged in size from 2209 nucleotides down to 102 nucleotides. All the deletion plasmids retained at least 36 nucleotides from the 5′-terminus and 51 nucleotides from the 3′-terminus of the DI genome. In cells expressing the five VSV proteins, the deleted DI RNAs were examined for their ability to be encapsidated, to replicate, and to bud to produce infectious DI particles. An RNA as small as 191 nucleotides, which contained 46 nucleotides from the 5′-end and 145 nucleotides from the 3′-end of the DI genome was encapsidated, replicated, and budded at least as efficiently as the full-length wild-type DI RNA. In contrast, a 102-nucleotide RNA that contained only the 51 nucleotides from the 5′-end of the DI RNA and its perfect 51-nucleotide complement at the 3′-end replicated poorly and failed to bud infectious DI particles. However, an RNA with an insertion of 1499-nucleotide “stuffer” sequences of non-VSV origin between the two 51-nucleotide complementary termini not only replicated but also budded infectious particles. These data show that the signals necessary for RNA encapsidation, replication, and packaging into infectious DI particles are contained within the 5′-terminal 36 nucleotides and the 3′-terminal 51 nucleotides of the DI RNA genome. Furthermore, the results show that a heterologous sequence can be replicated and packaged into infectious particles if it is flanked by the DI RNA termini.

Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

ABSTRACT To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.

Vesicular stomatitis virus RNA replication: a role for the NS protein

Synthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.

Bunyamwera Bunyavirus RNA Synthesis Requires Cooperation of 3′- and 5′-Terminal Sequences

ABSTRACT Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3′- and 5′-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3′ and 5′ NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3′- and 5′-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3′ and 5′ NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3′ and 5′ NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3′ and 5′ NTRs were selected.

Antigenic and Genetic Diversity among the Attachment Proteins of Group A Respiratory Syncytial Viruses That Have Caused Repeat Infections in Children

Antigenic differences between the two major groups of respiratory syncytial (RS) virus may contribute to reinfections with these viruses. Additional variability occurs within the two major groups; the importance of intra-group variability in reinfections with RS virus has not been defined. Two pairs of group A viruses that had caused sequential infections in children showed G protein amino acid differences of up to 15%. Vaccinia viruses were constructed that expressed the G proteins from 2 of the paired group A isolates. Immunization of cotton rats with the recombinant vaccinia viruses provided equal protection against intranasal challenge by either of the RS viruses. Despite the amino acid differences between the two group A RS virus G proteins, these animal studies did not reveal differences in protection after immunization with the two G proteins. Precise definition of the role of RS virus antigenic variability in the establishment of reinfections in humans will require further investigations in humans.

Recombinant vaccinia viruses expressing the F, G or N, but not the M2, protein of bovine respiratory syncytial virus (BRSV) induce resistance to BRSV challenge in the calf and protect against the development of pneumonic lesions.

The immunogenicity and protective efficacy of recombinant vaccinia viruses (rVV) encoding the F, G, N or M2 (22K) proteins of bovine respiratory syncytial virus (BRSV) were evaluated in calves, the natural host for BRSV. Calves were vaccinated either by scarification or intratracheally with rVV and challenged 6 to 7 weeks later with BRSV. Although replication of rVV expressing the F protein in the respiratory tract was limited after intratracheal vaccination, the levels of serum and pulmonary antibody were similar to those induced following scarification. The serum antibody response induced by the F protein was biased in favour of IgG1 antibody, whereas the G and the N proteins induced similar levels of IgG1:IgG2, and antibody was undetectable in calves primed with the M2 protein. The F protein induced neutralizing antibodies, but only low levels of complement-dependent neutralizing antibodies were induced by the G protein, and antibody induced by the N protein was not neutralizing. The F and N proteins primed calves for BRSV-specific lymphocyte proliferative responses, whereas proliferative responses were detected in calves primed with the G protein only after BRSV challenge. The M2 protein primed lymphocytes in only one out of five calves. Although there were differences in the immune responses induced by the rVVs, the F, G and N, but not the M2, proteins induced significant protection against BRSV infection and, in contrast with the enhanced lung pathology seen in mice vaccinated with rVV expressing individual proteins of human (H)RSV, there was a reduction in lung pathology in calves.

The Cys(3)-His(1) motif of the respiratory syncytial virus M2-1 protein is essential for protein function.

ABSTRACT The M2 gene of respiratory syncytial (RS) virus has two open reading frames (ORFs). ORF1 encodes a 22-kDa protein termed M2-1. The M2-1 protein contains a Cys3-His1 motif (C-X7-C-X5-C-X3-H) near the amino terminus. This motif is conserved in all human, bovine, and ovine strains of RS virus. A similar motif found in the mammalian transcription factor Nup475 has been shown to bind zinc. The M2-1 protein of human RS virus functions as a transcription factor which increases polymerase processivity, and it enhances readthrough of intergenic junctions during RS virus transcription, thereby acting as a transcription antiterminator. The M2-1 protein also interacts with the nucleocapsid protein. We examined the effects of mutations of cysteine and histidine residues predicted to coordinate zinc in the Cys3-His1 motif on transcription antitermination and N protein binding. We found that mutating the predicted zinc-coordinating residues, the cysteine residues at amino acid positions 7 and 15 and the histidine residue at position 25, prevented M2-1 from enhancing transcriptional readthrough. In contrast, mutations of amino acids within this motif not predicted to coordinate zinc had no effect. Mutations of the predicted zinc-coordinating residues in the Cys3-His1 motif also prevented M2-1 from interacting with the nucleocapsid protein. One mutation of a noncoordinating residue in the motif which did not affect readthrough during transcription, E10G, prevented interaction with the nucleocapsid protein. This suggests that M2-1 does not require interaction with the nucleocapsid protein in order to function during transcription. Analysis of the M2-1 protein in reducing sodium dodecyl sulfate-polyacrylamide gels revealed two major forms distinguished by their mobilities. The slower migrating form was shown to be phosphorylated, whereas the faster migrating form was not. Mutations in the Cys3-His1 motif caused a change in distribution of the M2-1 protein from the slower to the faster migrating form. The data presented here show that the Cys3-His1 motif of M2-1 is essential for maintaining the functional integrity of the protein.

Adding Genes to the RNA Genome of Vesicular Stomatitis Virus: Positional Effects on Stability of Expression

ABSTRACT Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.