Biomedical subjects

A Grakoui

Publications and source records attributed to A Grakoui.

21 records · Page 2Linked to original sources

PubMed1989

A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.

The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

Amino Acid Sequence↗

PubMed1989

Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4.

Temperature-sensitive (ts) mutants of Sindbis virus belonging to complementation group F, ts6, ts110, and ts118, are defective in RNA synthesis at the nonpermissive temperature. cDNA clones of these group F mutants, as well as of ts+ revertants, have been constructed. To assign the ts phenotype to a specific region in the viral genome, restriction fragments from the mutant cDNA clones were used to replace the corresponding regions of the full-length clone Toto1101 of Sindbis virus. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious transcripts, and the virus recovered was tested for temperature sensitivity. After the ts lesion of each mutant was mapped to a specific region of 400 to 800 nucleotides by this approach, this region of the cDNA clones of both the ts mutant and ts+ revertants was sequenced in order to determine the precise nucleotide change and amino acid substitution responsible for each mutation. Rescued mutants, which have a uniform background except for one or two defined changes, were examined for viral RNA synthesis and complementation to show that the phenotypes observed were the result of the mutations mapped. ts6 and ts110 had a single base substitution in nsP4, resulting in replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this, together with the fact that all RNA synthesis in ts6-infected cells and, to a lesser extent, in ts110-infected cells shut off when the cells were shifted from a permissive to a nonpermissive temperature, suggests that nsP4 is the virus polymerase. ts118 was a double mutant. It contained a single base substitution in nsP2, resulting in replacement of Val by Ala at position 425 that resulted in the formation of minute plaques, but not in a reduction in the plaque number at the nonpermissive condition. The second change, a substitution of Gln by Arg in ts118 at residue 93 in nsP4, had little apparent phenotype on its own, but in combination with the change in nsP2 led to a ts phenotype. Thus, in each case the mutation responsible for the temperature sensitivity of the three known complementation group F mutants lay in nsP4. In addition, the result with ts118 suggests that nsP2 and nsP4 may interact with each other in a complex.

Amino Acid Sequence↗

PubMed1989

Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation.

Yellow fever (YF) virus is the prototype member of the flavivirus family, a diverse group of human and animal pathogens. A live-attenuated strain of YF virus, called 17D, has been used successfully for human vaccination for more than 50 years. In this report we describe the construction of full-length YF 17D cDNA templates that can be transcribed in vitro to yield infectious YF virus RNA. Because of the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, we developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. The YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria. This system should facilitate the molecular genetic analysis of flavivirus replication and attenuation and may allow YF 17D to be used as a carrier for immunologically important epitopes from other disease agents.

Base Sequence↗