Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

The viral RNA 3′- and 5′-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.     

Nucleotide sequence analysis of six satellite RNAs of cucumber mosaic virus: primary sequence and secondary structure alterations do not correlate with differences in pathogenicity

The nucleotide sequences of six satellite RNAs of cucumber mosaic virus (CMV) differing in their pathological properties have been determined. Although these RNAs varied in length from 333 to 342 nucleotides, the extent of nucleotide sequence homology to each other and to four other satellite RNAs ranged from 84 to 97%, allowing satellite RNA subgrouping on the basis of nucleotide sequence. This subgrouping was further extended by an analysis of the nature of the differences in nucleotide sequence, with most of the variation occurring within nucleotides 129 to 190 and 318 to the 3'-end. Enzymatic and chemical secondary structure probing of four satellite RNAs yielded a common structure in which 48-52% of the nucleotides were base paired. Although there were discrepancies between the experimental data and the model in two out of seven hairpins, the model contained structural elements and consensus sequences in common with the 3'-end sequences of two satellite RNA helper viruses: CMV and tomato aspermy virus. Specific sequence changes, secondary structure characteristics, or proteins potentially encoded by any of 10 satellite RNAs did not correlate with the known pathogenic responses attributable to individual satellite RNAs.     

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.     

Detection of small RNAs containing the 5'- and the 3'-end sequences of viral genome during West Nile virus replication

In the first step of flavivirus replication, the 5'-end of viral genomic RNA is thought to interact with the 3'-end of the genomic RNA at the complimentary sequences (CSs) located at both ends of the genomic RNA. However, there is little evidence of direct interaction between the two ends of the viral genomic RNA in virus-replicating cells. Herein, we show that viral small negative-strand RNA species, composed of two ends corresponding to the upstream of the 5'-end CS and the downstream of the 3'-end CS of viral genomic RNA, were synthesized during viral replication. We hypothesized that the viral small negative-sense RNAs were synthesized during viral negative-sense RNA synthesis through the template-jumping of viral RNA-dependent RNA polymerase from the 3'-end to the 5'-end of viral genomic RNA used as a template. Our present results strongly indicate that the two ends of viral genomic RNA associate with each other during viral replication.     

Sequence analysis of the 5′ ends of tomato spotted wilt virus N mRNAs

Summary Messenger RNAs transcribed from the tomato spotted wilt virus (TSWV) RNA genome have characteristic extra non-templated heterogeneous sequences at their 5 ends which may be the result of a cap-snatching event involving cellular mRNAs. In order to investigate the genetic origin of these extra sequences and to gain more insight in the process of cap-snatching as performed by TSWV, nucleocapsid protein (N) mRNAs derived from the TSWV S RNA were cloned and sequenced. Twenty clones were obtained which contained 5-proximal sequences of non-viral origin, ranging in length from 12 to 21 nucleotides. None of the sequences analyzed were identical and no base preference at the endonucleolytic site was observed.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.