Basic amino acid residues at the carboxy-terminal eleven amino acid region of the phosphoprotein (P) are required for transcription but not for replication of vesicular stomatitis virus genome RNA.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein N in vivo. We have shown that the deletion of 11 amino acids from the C terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesis in vitro in a transcription reconstitution reaction as well as transcription in vivo of a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) both in vitro and in vivo, suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N protein in vivo, indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNA in vivo. Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively.

Inhibition of vesicular stomatitis virus in cells constitutively expressing an antisense RNA targeted against the virus RNA polymerase gene.

To study the effect of virus-specific antisense RNA expression on vesicular stomatitis virus (VSV) infectivity in cultured cells, a HeLaS3 cell line constitutively expressing antisense RNA complimentary to a portion of the VSV large RNA-dependent RNA polymerase gene (L) was established (HeAntiL). At an m.o.i. of 0.01 or 0.1, the HeAntiL cell line was able to reduce virus titre and delay virus-induced cell death by 9 or 5 h, respectively, when compared to a HeLa cell line stably transfected with the expression vector devoid of antisense sequence. Ribonuclease protection experiments showed a 10-20-fold reduction of hybridizable virus L mRNA in infected HeAntiL cells compared to infected control cells at various times before cell death. These results indicate that the antisense RNA approach can significantly reduce VSV mRNA transcription and virus production for a reasonable period of time. The robust growth rate of VSV eventually overwhelms the available antisense RNA and leads to delayed cell death.

Efficient interaction of the vesicular stomatitis virus P protein with the L protein or the N protein in cells expressing the recombinant proteins.

Specific in vivo interaction between the phosphoprotein (P) and the large polymerase protein (L) from the Indiana serotype of vesicular stomatitis virus was studied using a two-hybrid system. Transfection of CHO cells with plasmids encoding GALPIND and VPLIND fusion proteins resulted in an easily detectable level of CAT activity, indicating that PIND and LIND associate in vivo in the absence of other viral proteins. Mutational studies of PIND demonstrated that both domains I and II of PIND are important for PIND-LIND association. In addition, casein kinase II (CKII)-mediated phosphorylation within domain I of PIND was necessary for efficient association with LIND. We have also used the two-hybrid system to show PIND interaction with NIND in vivo. PIND and NIND associated more strongly than PIND and LIND. A similar strong association was observed in heterologous interaction studies between Indiana and New Jersey serotype P and N proteins. Mutational studies of PIND demonstrated that, unlike what was found for PNJ-NNJ association, only the C-terminal region of the P protein was important for efficient association with NIND. Like PNJ, CKII-mediated phosphorylation within domain I of PIND was not required for P-N association and, like NNJ, the C-terminal five amino acids of the NIND protein were critical for P association with N. These results demonstrate the importance of phosphorylation and specific domains of the P protein in its interaction with the L and N proteins, which are necessary for viral transcription and replication, respectively.

Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system.

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.

Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro.

The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphoprotein and the crucial role played by a cell protein kinase in this process.

Alteration of specific amino acid residues in the acidic domain I of VSV phosphoprotein (P) converts a GAL4-P(I) hybrid into a transcriptional activator.

As part of a study of transcriptional regulation by viral proteins, we examined whether an acidic region from a regulatory protein of an RNA virus could function as a trans-activator. The NH2-terminal highly acidic domain I of the phosphoprotein (P) of vesicular stomatitis virus (VSV) was fused to the DNA-binding domain of the yeast trans-activator, GAL4. In transient transfection assays, the resulting chimeric protein failed to activate transcription of a reporter CAT gene. However, mutation of basic amino acid residues located at positions 6 and 8 or the alteration of eight amino acids within the acidic domain to eight different amino acids converted the chimeric protein into a transcriptional activator comparable to wild-type GAL4. When subjected to SDS-polyacrylamide gel electrophoresis, the P proteins containing trans-activation-positive mutations in domain I showed an altered mobility, suggesting that these mutations may have caused a conformational change that is critical for trans-activation. Since the acidity of P domain I is not sufficient to activate transcription, additional features of this region must play an important role in GAL4-mediated trans-activation. None of the trans-activation-positive mutants supported VSV RNA transcription in vitro. These results suggest that the amino acid residues within P domain I that can be made to function in the trans-activation of DNA-dependent RNA transcription are distinct from those involved in VSV RNA-dependent RNA transcription.

Organization and expression of 5S rRNA genes in the parasitic nematode, Brugia malayi.

DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.

A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.

The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.