Lisa H. Soe

Primary structure and translation of a defective interfering rna of murine coronavirus

Abstract An intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DIssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the Fend, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3′ end of the parental MHV genome. The DIssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DI-infected cells. Sequence comparison of DIssE and the corresponding parts of the parental virus genome revealed that DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5′ end of DIssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.

The 5′-end sequence of the murine coronavirus genome: Implications for multiple fusion sites in leader-primed transcription

Abstract The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5′-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5′-end of the mouse hepatitis virus genomic RNA. The 5′-terminal sequences are identical to the leader sequences present at the 5′-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3′-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5′-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7–18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

Genetic analysis of the feline RD-114 retrovirus-related endogenous elements

The structure of the multiple copies of the endogenous RD-114 retroviral sequences in the domestic cat were compared by restriction mapping of cloned and genomic DNAs. Six different clones, each representing a different loci, were randomly chosen from 20 clones isolated from a cat DNA library. Whereas all clones contained viral structural sequences which were extensively homologous to the gag and pol regions of exogenous RD-114, they varied from each other in the extent of deletions (0.7-1.9 kilobase pairs) in the env region. Each clone contained a pair of LTRs which bracketed the structural genes, implicating an origin from exogenous infection of a distant ancestor of the domestic cat. Analysis of genomic DNA from six separate domestic cats also indicated that the majority of the RD-114 endogenous loci contained variably deleted env and conserved gag-pol regions. The occurrence of at least 13 unique RD-114-related loci could be detected in each cat DNA examined. It appeared that among these multiple copies inherited in the cat genome, there is only a single copy which would correspond to the undeleted RD-114 provirus, and which may represent the locus for the inducible RD-114 virus.

Structure of the polymorphic feline c-myc oncogene locus

Two feline c-myc DNA clones (CM-2 and CM-3), isolated from a cat DNA library, are structurally very similar. However, they differ at a SmaI site in exon III which is present only in CM-2. In the outbred feline population, cats heterozygous for this site or homozygous for the CM-3-type gene have been observed. The results provide a physical map of the feline c-myc locus, and define hitherto unidentified alleles of this gene.

Murine coronavirus 5'-end genomic RNA sequence reveals mechanism of leader-primed transcription.

Mouse hepatitis virus (MHV) contains a single-strand, positive-sense RNA genome which is transcribed in infected cells, first, into a full-length negative-strand RNA (Brayton et al., 1982; Lai et al., 1982) and then into a positive-sense genomic RNA and six species of subgenomic mRNA. The mRNAs consist of a 3′ co-terminal nested-set structure (Lai et al., 1981), and also contain an identical leader sequence of approximately 72 nucleotides at the 5′ ends (Lai et al., 1984; Spaan et al., 1983). Ultraviolet transcriptional mapping studies (Jacobs et al., 1981) and the fact that no nuclear function is required for replication (Brayton et al., 1981; Wilhelmsen et al., 1981) suggest that the joining of the leader sequences to coronavirus mRNAs does not utilize conventional eukaryotic splicing mechanisms.