The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5-cap structures methylated at the guanine-N7 and 2-O-adenosine positions (7mGpppA m ). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the Lpolymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-L-methionine-binding domain of the L protein.Vesicular stomatitis virus (VSV, a rhabdovirus) is a prototypic nonsegmented negative-strand (NNS) RNA virus belonging to the order Mononegavirales, whose members share a similar genome organization and common mechanisms of genome replication and gene expression. This order includes many medically important pathogens, including the lethal rabies, Ebola, Marburg, Nipah, and Hendra viruses. The RNA-dependent RNA polymerase (RdRp) of NNS RNA viruses is packaged into mature virions and consists of two viral subunits, the phosphoprotein (P) and the large (L) protein. The RNA genome of NNS viruses is tightly encapsidated by the nucleocapsid (N) protein, and the resulting nucleocapsid serves as the template for the sequential transcription of monocistronic mRNAs and for genome replication. Recent studies on VSV suggest that two separate RdRp complexes, which differ in their protein content, are involved in genome replication versus mRNA transcription (14, 47).The VSV RdRp produces mRNA transcripts modified at their 5Ј end by capping and cap methylation (71). The mechanism of mRNA 5Ј capping in VSV and other NNS RNA viruses is unusual, where, in contrast to cellular cap structures, both the ␣ and ␤ phosphates in the GpppA triphosphate bridge are derived from a GDP donor (2, 5, 23). The cytoplasmic localization of virus transcription and the unusual mechanism of capping suggest that the guanylyltransferas...

No abstract

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated alpha-chain associated with an alpha + thalassaemia phenotype. The amount of alpha mRNA relative to beta mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of alpha + thalassaemia. In a patient with Hb CS-H disease there was greater alpha/beta mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the alpha mRNA in the patient's peripheral blood was derived from the alpha 1 (alpha A) gene. The data suggest that alpha CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3' non-coding region during translation.

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.