Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity.

The diazotrophic bacteria collectively known as "rhizobia" are important for establishing symbiotic N(2)-fixing associations with many legumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptable yields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNA gene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis (MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12 Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK, gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common bean ranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common bean also formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutual agreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broad genetic diversity among strains classified as Rhizobium tropici, providing evidence of new species.

Immunological identification of Tacaribe virus proteins.

Tacaribe virus (TV), an arenavirus, is an enveloped virus with genetic information encoded in two segments of single-stranded RNA. The completed sequence of TV led to the identification of four open reading frames (ORF). In order to establish a direct link between ORFs in the sequence of TV and proteins present in virus particles and virus-infected cells, segments of the molecularly cloned TV genome were engineered so as to be expressed in Escherichia coli to produce fusion proteins that were used to raise antisera. The antisera were in turn employed to identify the TV gene products. Serum to the putative nucleocapsid (N) protein reacted with a 68-kDa protein, both in TV particles and in the infected cells. Sera raised to the glycoprotein precursor (GPC) immunoprecipitated two proteins of 68 and 70 kDa from infected cell lysates. Analysis of GPC synthesis in the presence of tunicamycin revealed that the unglycosylated GPC appeared as two polypeptides of 43 and 46 kDa. The putative RNA polymerase gene product (L) was detected as a approximately 240-kDa protein. Serum to the small zinc-binding domain protein (p11-Z) recognized a protein of approximately 11kDa. Immunological evidence is presented that in addition to N and L, two glycoproteins (GP1 and GP2) and p11-Z are structural components of Tacaribe virions.

Expresión como proteínas de fusión de las subunidades PB1 y PB2 de la polimerasa del virus de la influenza / Expression how proteins of fusion of the subunits PB1 and PB2 of the influenza viral of polymerase

Los cADNs de los genes que codifican las proteínas PB1 y PB2 de la ARN polimerasa del virus de la influenza, se clonaron en fase en un sitio distal del ATG que codifica la proteína 10 del bacteriógafo T7, en el vector de expresión pAR3040, bajo el control de un promotor de la ARN polimerasa de T7. Los plásmidos recombinantes se introdujeron en la cepa bacteriana. E coli BL21 (DE3)plysS, que contiene en su genoma el gen de la ARN polimerasa del fago T7, bajo el control de un promotor (lac uv5) inducible por IPTG. La inducción de la ARN polimerasa del fago T7 en las células hospederas, resultó en la expresión de las proteínas PB1 y PB2 en forma de proteínas de fusión. Las proteínas expresadas se acumularon en grandes cantidades en el interior de las células bacterianas y fueron recuperadas de los lisados en forma de precipitados insolubles.