Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form.

Second-site mutations selected in transcriptional regulatory sequences compensate for engineered mutations in the vesicular stomatitis virus nucleocapsid protein.

The active template for RNA synthesis for vesicular stomatitis virus (VSV) and other negative-strand viruses is the RNA genome in association with the nucleocapsid (N) protein. The N protein molecules sequester the genomic RNA and are linked together by a network of noncovalent interactions. We previously demonstrated that mutations predicted to weaken interactions between adjacent N protein molecules altered the levels of RNA synthesis directed from subgenomic ribonucleoprotein (RNP) templates.

Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells.

Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored.

Helical virus structure: the case of the rhabdovirus bullet.

Commentary on Ge, P.; Tsao, J.; Schein, S.

Importance of hydrogen bond contacts between the N protein and RNA genome of vesicular stomatitis virus in encapsidation and RNA synthesis.

Vesicular stomatitis virus (VSV) genomic RNA encapsidated by the nucleocapsid (N) protein is the template for transcription and replication by the viral polymerase. We analyzed the 2.9-A structure of the VSV N protein bound to RNA (T.

Mutations in the C-terminal loop of the nucleocapsid protein affect vesicular stomatitis virus RNA replication and transcription differentially.

The 2.9-A structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template.

A temperature sensitive VSV identifies L protein residues that affect transcription but not replication.

To investigate the polymerase components selectively involved in transcription versus replication of vesicular stomatitis virus (VSV), we sequenced the polymerase gene of a conditionally RNA defective, temperature sensitive VSV: ts(G)114, which has a phenotype upon shift from permissive to non-permissive temperature of shut-down of mRNA transcription and unaffected genome replication. Sequence analysis of the ts(G)114 L gene identified three altered amino acid residues in the L protein. These three changes were specifically engineered individually and in combinations into a functional cDNA clone encoding the VSV genome and tested for association with the temperature sensitive and RNA defective phenotypes in the background of recovered engineered viruses.

Analysis of a structural homology model of the 2'-O-ribose methyltransferase domain within the vesicular stomatitis virus L protein.

The large (L) proteins of non-segmented negative stranded (NNS) RNA viruses contain the core RNA dependent RNA polymerase activity for RNA replication and transcription as well as the activities for polyadenylating and capping the mRNA transcripts and for methylating the cap structures. There is currently no structural information available for these large multi-functional proteins. Phylogenetic analyses have led to the division of the L protein primary structure into six functional domains of high conservation that are linked by variable regions.

S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein.

There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3' end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5' cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM.

Selection for gene junction sequences important for VSV transcription.

The heptauridine tract at each gene end and intergenic region (IGR) at the gene junctions of vesicular stomatitis virus (VSV) have effects on synthesis of the downstream mRNA, independent of their respective roles in termination of the upstream mRNA. To investigate the role of the U tract and the IGR in downstream gene transcription, we altered the N/P gene junction of infectious VSV such that transcription levels would be affected and result in altered molar ratios of the N and P proteins, which are critical for optimal viral RNA replication. The changes included extended IGRs between the N and P genes and shortening the length of the heptauridine tract upstream of the P gene start.

Human respiratory syncytial virus glycoproteins are not required for apical targeting and release from polarized epithelial cells.

Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release.

The VSV polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation.

Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene.

The stability of human respiratory syncytial virus is enhanced by incorporation of the baculovirus GP64 protein.

Current efforts to develop a vaccine against human respiratory syncytial virus (HRSV) are focused on live attenuated strains. However, the unstable nature of HRSV is a major challenge for the preparation, storage and distribution of live vaccine candidates. We report here that the stability of HRSV can be improved by incorporation of the GP64 glycoprotein from baculovirus Autographa californica multiple nucleopolyhedrovirus.

The cytoplasmic tail of the human respiratory syncytial virus F protein plays critical roles in cellular localization of the F protein and infectious progeny production.

The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2).

Structure of the vesicular stomatitis virus nucleoprotein-RNA complex.

Vesicular stomatitis virus is a negative-stranded RNA virus. Its nucleoprotein (N) binds the viral genomic RNA and is involved in multiple functions including transcription, replication, and assembly. We have determined a 2.

Identification of the Bunyamwera bunyavirus transcription termination signal.

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3' ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5' terminus of the genomic template.

The Bunyamwera virus mRNA transcription signal resides within both the 3' and the 5' terminal regions and allows ambisense transcription from a model RNA segment.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of RNA viruses. BUNV genomic strands are templates for both replication and transcription, whereas the antigenomic RNAs serve only as templates for replication. By mutagenesis of model templates, we showed that the BUNV transcription promoter comprises elements within both the 3' and the 5' nontranslated regions.

Role of the conserved nucleotide mismatch within 3'- and 5'-terminal regions of Bunyamwera virus in signaling transcription.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3' and 5' nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable.

Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: identification of amino acid residues modulating pH-dependent infectivity.

We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ.

Respiratory syncytial virus and other pneumoviruses: a review of the international symposium--RSV 2003.

The Respiratory Syncytial Virus 2003 symposium took place from 8th-11th November 2003 in Stone Mountain, Georgia, and brought together more than 200 international investigators engaged in RSV research. RSV biology, pathogenesis, and clinical data, as well as RSV vaccines and antivirals, were addressed in the meeting, and this review will aim to briefly summarize and discuss the implications of new findings. The meeting also served as the inauguration of the Robert M.

Recombinant vesicular stomatitis (Indiana) virus expressing New Jersey and Indiana glycoproteins induces neutralizing antibodies to each serotype in swine, a natural host.

Vesicular stomatitis virus (VSV) is the most common cause of vesicular disease outbreaks in livestock throughout the Western Hemisphere. Two major serotypes, Indiana and New Jersey, cause epidemic disease in pigs, cattle, and horses. We generated recombinant viruses derived from the Indiana serotype genome that were engineered to contain and express: (1) a single copy of the glycoprotein gene from the Indiana serotype (VSIV-GI); (2) a single copy of the glycoprotein gene from the New Jersey serotype (VSIV-GNJ); or (3) two copies of the glycoprotein gene, one from each of the two major VSV serotypes (VSIV-GNJGI) [Martinez I, Rodriguez LL, Jimenez C, Pauszek SJ, Wertz GW.

Fitness analyses of vesicular stomatitis strains with rearranged genomes reveal replicative disadvantages.

Gene expression of the nonsegmented negative-strand RNA viruses is determined by the position of each gene relative to that the single 3' promoter. The general order of genes among all of the viruses of the order Mononegavirales is highly conserved. In previous work we generated recombinant viruses in which the order of the three central genes of the prototypical rhabdovirus, vesicular stomatitis virus, was rearranged to all six possible permutations.

Variations in intergenic region sequences of Human respiratory syncytial virus clinical isolates: analysis of effects on transcriptional regulation.

Sequences at the beginnings and ends of Human respiratory syncytial virus (HRSV) genes are necessary for efficient initiation and termination of transcription. The gene start sequences are well conserved and contain signals required for initiation, while the semi-conserved sequences at the gene ends direct transcriptional termination with varying efficiencies. The intergenic regions, which lie between the gene ends and the downstream gene start sequences, are not conserved in length or sequence, and certain positions have been reported to play a role in transcriptional regulation.

trans-Complementation allows recovery of human respiratory syncytial viruses that are infectious but deficient in cell-to-cell transmission.

Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported.

Antigenic and genetic variation in human respiratory syncytial virus.

Background: Human respiratory syncytial virus (HRSV) is a leading cause of serious pediatric respiratory disease worldwide. Natural infection provides only partial protection as repeat infections occur throughout life. A brief review of the extent of antigenic and genetic variation observed in HRSV clinical isolates is presented.