James G. Keck

A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus.

Abstract Coronaviruses undergo RNA recombination at a very high frequency. To understand the mechanism of recombination in murine coronavirus, we have performed RNA sequencing of viral genomic RNA to determine the precise sites of recombination in a series of recombinants which have crossovers within the gene encoding the peplomer protein. We found that all of the recombination sites are clustered within a region of 278 nucleotides in the 5′-half of the gene. This region in which all of the crossovers occurred represents a small fraction of the distance between the two selection markers used for the isolation of these recombinant viruses. This result suggests that this region may be a preferred site for RNA recombination. The crossover sites are located within and immediately adjacent to a hypervariable area of the gene. This area has undergone deletions of varying sizes in several virus strains which have been passaged either in vivo or in vitro. These results suggest that a similar RNA structure may be involved in the occurrence of both recombination and deletion events.

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

Abstract Mouse hepatitis virus (MHV), a murine coronavinus, 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.

Defective-interfering particles of murine coronavirus: Mechanism of synthesis of defective viral RNAs

Abstract The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interfering (DI) particles of mouse hepatitis virus was studied. Two DI-specific RNA species, DIssA of genomic size and DIssE of subgenomic size, were detected in DI-infected cells. Purified DI particles, however, were found to contain predominantly DIssA and only a trace amount of DIssE RNA. Despite its negligible amount, the DIssE RNA in virions appears to serve as the template for the synthesis of DIssE RNA in infected cells. This conclusion was supported by two studies. First, the uv target size for DIssE RNA synthesis is significantly smaller than that for DIssA. Second, when purified DIssE RNA was transfected into cells which had been infected with a helper virus, DIssE RNA could replicate itself and became a predominant RNA species in the infected cells. Thus, DIssE RNA was not synthesized from the genomic RNA of DI particles. By studying the relationship between virus dilution and the amount of intracellular viral RNA synthesis, we have further shown that DIssE RNA synthesis requires a helper function, but it does not utilize the leader sequence of the helper virus. In contrast, DIssA synthesis appears to be helper-independent and can replicate itself. Thus DIssA codes for a functional RNA polymerase.

Temporal regulation of bovine coronavirus RNA synthesis.

Abstract The structure and synthesis of bovine coronavirus (BCV)-specific intracellular RNA were studied. A genome-size RNA and seven subgenomic RNAs with molecular weights of approximately 3.3 × 106, 3.1 × 106, 2.6 × 106, 1.1 × 106, 1.0 × 106, 0.8 × 106 and 0.6 × 106 were detected. Comparisons of BCV intracellular RNAs with those of mouse hepatitis virus (MHV) demonstrated the presence of an additional RNA for BCV, species 2a, of 3.1 × 106 daltons. BCV RNAs contain a nested-set structure similar to that of other coronaviruses. This nested-set structure would suggest that the new RNA has a capacity to encode a protein of approximately 430 amino acids. Kinetic studies demonstrated that the synthesis of subgenomic mRNAs and genomic RNA are differentially regulated. At 4 to 8 h post-infection (p.i.), subgenomic RNAs are synthesized at a maximal rate and represent greater than 90% of the total viral RNA synthesized, whereas genome-size RNA accounts for only 7%. Later in infection, at 70 to 72 h p.i., genome-size RNA is much more abundant and accounts for 88% of total RNA synthesized. Immunoprecipitations of [35S]methionine-pulse-labeled viral proteins demonstrated that viral protein synthesis occurs early in the infection, concurrent with the peak of viral subgenomic RNA synthesis. Western blot analysis suggests that these proteins are stable since the proteins are present at high level as late as 70 to 72 h p.i. The kinetics of production of virus particles coincides with the synthesis of genomic RNA. These studies thus indicate that there is a differential temporal regulation of the synthesis of genomic RNA and subgenomic mRNAs, and that the synthesis of genomic RNA is the rate-limiting step regulating the production of virus particles.

Inhibition of murine coronavirus rna synthesis by hydroxyguanidine derivatives

Abstract A series of hydroxyguanidine derivatives, which are substituted salicylaldehyde Schiff-bases of 1-amino-3- hydroxyguanidine tosylate, were tested for the inhibition of RNA synthesis of mouse hepatitis virus (MHV). It was shown that these compounds could selectively inhibit virus-specific RNA synthesis. Every aspect of viral RNA synthesis, including synthesis of negative-stranded RNA, subgenomic mRNA transcription and genomic RNA replication, was inhibited to roughly the same extent. These compounds are the first known inhibitors of coronaviral RNA synthesis and should prove useful for understanding the mechanism of viral RNA synthesis.

Temporal Regulation of RNA Synthesis of Bovine Coronavirus

It has been shown with mouse hepatitis virus (MHV) and avian infectious bronchitis virus that all of the subgenomic mRNAs and genomic RNA are synthesized at the same rate throughout the viral replication cycle (Leibowitz et al., 1981; Stern and Kennedy, 1980). The viral genomic RNA gradually accumulates late in infection. However, no clear switching from transcription of subgenomic mRNAs to replication of genomic-sized RNA is seen. Thus, it is not known whether there is a temporal regulation of viral RNA synthesis in coronavirus-infected cells.

Characterization of Monoclonal Antibodies to Human OC43

Human coronaviruses are important respiratory pathogens (1). These viruses have also been implicated in human neurological diseases (2, 3), and some reports have suggested an association with the demyelinating condition multiple sclerosis (4, 5, 6), although other studies cast doubt upon the role of coronaviruses in this disease (7, 8, 9, 10, 11). It has been shown that most human coronaviruses belong to one of two antigenic groups represented by isolates OC43 or 229E respectively (1, 12). The antigenic properties of these coronaviruses are thus of medical and virological interest.