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At least 127 records · Page 7Linked to original sources
PubMed1988

Rift Valley fever virus M segment: cell-free transcription and translation of virus-complementary RNA.

A cell-free system has been used to study gene expression of the M segment RNA of the Phlebovirus Rift Valley fever virus (RVFV). RVFV sequence-containing plasmids were used to synthesize M segment mRNA-like transcripts. These transcripts were then translated in vitro in the absence or presence of microsomal membranes. Cell-free translation of a transcript which closely resembled authentic M segment mRNA (RNA-7) yielded a primary translation product of 133 kilodaltons (kDa), the size expected of a polypeptide encompassing the entire open reading frame (ORF) of the M segment. When translations were conducted in the presence of microsomal membranes, this primary protein was cotranslationally processed to yield the two viral glycoproteins, G1 and G2, as well as proteins of 78, 21, and 14 kDa. With one exception, these in vitro processed polypeptides comigrated with M segment-encoded proteins found in RVFV-infected cell lysates. A polypeptide corresponding to the in vitro 21-kDa protein was not detected in vivo. To investigate translational initiation and processing of the protein products of the M segment, additional transcripts were generated in which varying portions of the amino-terminal "preglycoprotein" region of the M segment ORF were deleted. Translation results indicated that the 78- and 21-kDa proteins were initiated from the first methionine codon of the ORF, and the 14-kDa polypeptide began from the second in-phase ATG. These products and a major portion of the preglycoprotein region sequence were not required for the proper synthesis and processing of the viral glycoproteins in vitro. In light of these results, possible expression strategies used by this Phlebovirus M segment RNA are discussed.

Bunyaviridae↗
PubMed1998

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Base Sequence↗
PubMed1983

Nonviral heterogeneous sequences are present at the 5' ends of one species of snowshoe hare bunyavirus S complementary RNA.

Analyses of the 5' ends of snowshoe hare bunyavirus plus sense S RNA species (including mRNA) recovered from infected cells have revealed two types of termini. These include ends that are essentially exact copies of the 3' end of the viral S RNA and others that are similar, but additionally have 13-14 nucleotide extensions that are heterogeneous in sequence. The former probably represent replicative plus sense RNA species, the latter mRNA species that have host cell derived primer sequences.

Animals↗
PubMed1977

Suitability of Epstein-Barr virus DNA obtained from superinfected Raji cells for complementary RNA hybridization studies.

A method was established to obtain a high yield of Epstein-Barr virus (EBV) DNA for nucleic acid hybridization studies on latent virus DNA in transformed cells. Superinfection of Raji cells with EBV concentrated from HRI cell cultures produced a 600-fold higher yield of EBV DNA than direct isolation of EBV from HRI cell cultures. The virus DNA thus prepared from Raji cells superinfected with EBV was radioisotopically and spectrophotometrically pure and served as a satisfactory template for the preparation of cRNA specific to EBV DNA.

Burkitt Lymphoma↗
PubMed1983

Identification of four complementary RNA species in Akabane virus-infected cells.

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 X 10(6), 1.5 X 10(6), and 0.48 X 10(6) for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind to oligodeoxythymidylate-cellulose. Kinetic analysis of cRNA synthesis in infected cells at various times postinfection suggested that cRNA synthesis could be detected as early as 2 h postinfection and that maximal synthesis occurred at 4 to 6 h postinfection. The RNAs synthesized in infected cells could be partially resolved by sucrose density gradient centrifugation. The RNA fraction that cosedimented with the S segment of viral genomic RNA yielded two duplex RNA species when hybridized with viral genomic RNA, suggesting the presence of two small cRNA species. Specific hybridization with individual viral genomic RNAs confirmed that two species of cRNA are coded by the S RNA segment. Analysis of cRNA synthesis in the presence of the protein synthesis inhibitors cycloheximide and puromycin indicated that cycloheximide completely inhibited virus-specific RNA synthesis early and late in infection, whereas a very low level of synthesis occurred in the presence of puromycin. The inhibitory effects of these drugs were found to be reversible when the drugs were washed from the cells. It is concluded that continued protein synthesis is required for cRNA synthesis to proceed in Akabane virus-infected cells.

Animals↗