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.     

Expression of the peplomer glycoprotein of murine coronavirus JHM using a baculovirus vector

The gene encoding the E2 peplomer glycoprotein of coronavirus mouse hepatitis virus JHM strain (JHMV) has been inserted into the genome of Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the coding region of the AcNPV polyhedrin gene. This recombinant virus produced E2 protein in insect cells under the control of the baculovirus polyhedrin promotor. The expressed E2 protein was shown in size and antigenic properties to be similar to the E2 protein produced in mouse cells infected by JHMV. The expressed E2 protein was glycosylated and transported to the cell surface; however, no proteolytic cleavage was detected in insect cells. The sera from rats immunized with partially purified E2 protein derived from insect cells reacted in immunoprecipitation and immunofluorescence experiments with the E2 protein produced in JHMV-infected mouse cells. The antiserum failed to neutralize the infectivity of JHMV. These results suggest that the E2 protein expressed by the recombinant baculovirus in insect cells is similar but not identical to the E2 protein produced in JHMV-infected mouse cells. The inability of the E2 protein expressed in insect cells to produce neutralizing antibody is discussed.     

A region of heterogeneity adjacent to the 5s ribosomal RNA gene of cereal rusts

Summary Total genomic DNA was isolated from three cereal stem rusts, Puccinia graminis f. sp. tritici, f. sp. secalis, f. sp. avenae, and two cereal leaf rusts, P. recondita f. sp. tritici and P. coronata f. sp. avenae, and analyzed for the presence of heterogeneity in the intergenic region of the ribosomal DNA repeat unit. A 1 kb region of the repeat unit between the 26s and the 5s rRNA genes (IGR-1) was amplified by PCR and was found to be heterogeneous within each isolate and variable in size between races and species. The PCR results were confirmed by Southern blot analysis of native DNA. In an isolate of race C36(48), heterogeneity appeared to be due to variable numbers of 0.1 kb subrepeats in IGR-1. Nine wheat stem rust strains representing nine different races produced a unique pattern of heterogeneity while two different isolates of one race were identical, as were five of another. This may provide a rapid method for race identification in wheat stem rust. Heterogeneity and polymorphism in rye stem rust, oat stem rust, wheat leaf rust, and oat crown rust, was less pronounced than in wheat stem rust. In the course of this work, the 5s rRNA gene was located and its position and orientation within the ribosomal repeat unit was established.     

Localization of major neutralizing epitopes on the S1 polypeptide of the murine coronavirus peplomer glycoprotein.

A recombinant baculovirus system has been used to express the amino terminal half of the murine coronavirus (JHMV) peplomer glycoprotein in insect cells. The expressed polypeptide is glycosylated and is recognized by a set of monoclonal antibodies (mAbs) specific for JHMV S protein. Three of these mAbs have a very high neutralizing activity for JHMV but not for other MHV strains. These results indicate that JHMV-specific, major neutralizing epitopes reside in the amino terminal S1 subunit of the peplomer glycoprotein.     

Genomic RNA of the murine coronavirus JHM.

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.     

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

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 5' end, 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.     

Random nature of coronavirus RNA recombination in the absence of selection pressure.

RNA-RNA recombination is thought to occur preferentially at certain selected sites and in only a few RNA viruses; the mechanism for these restrictions is unknown. In this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. Thus, coronavirus RNA recombination appears to be more random than previously realized. However, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported "hot spot" for coronavirus recombination. These results suggest that RNA recombination is common and random in nature, but only certain recombinants can be selected. Thus, the presence of recombinational "hot spots" for coronavirus or other RNA viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of RNA recombination. The failure to detect recombinants in other RNA viruses may therefore be due to unfavorable properties of recombinant viruses. This approach can be used to detect recombinants in these viruses.     

Sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity.

Mouse hepatitis virus (MHV), a coronavirus, causes encephalitis and demyelination in susceptible rodents. Previous investigations have shown that the MHV spike (S) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. To understand the molecular basis of MHV neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the JHM strain of MHV, which were selected with monoclonal antibodies (MAbs) specific for the S protein. We found that variant 2.2-V-1, which was selected with MAb J.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (NT) 3340, as compared to the parental JHM virus, which predominantly caused encephalitis. This site was in the S2 subunit of the S protein. In contrast, variant 7.2-V-1, which was selected with MAb J.7.2 and primarily caused encephalitis, had two point mutations at NT 1766 and 1950, which were in the S1 subunit. Finally, the double mutant 2.2/7.2-V-2, which was selected with both MAbs J.2.2 and J.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from NT 1523 to 1624 in the S1 and a point mutation at NT 3340 in the S2. We conclude that at least two regions of the S protein contribute to neuropathogenicity of MHV. We have also isolated a partial revertant of 2.2-V-1, which was partially resistant to MAb J.2.2 but retained the same neuropathogenicity as the variant 2.2-V-1. This revertant retained the mutation at NT 3340, but had a second-site mutation at NT 1994, further confirming that NT 3340 contributed to the pathogenic phenotype of MHV. By comparing these results with MHV variants isolated in other laboratories, which had mutations in other sites on the S gene and yet retained the demyelinating ability, we suggest that the ability of JHM viruses to induce demyelination is determined by the interaction of multiple sites on the S gene, rather than the characteristics of a single, unique site. Our study also revealed the possible presence of microheterogeneity of S gene sequence, particularly in the S1 region, in these viruses. The sequence microheterogeneity may also contribute to the differences in their biological properties.     

Mapping recombination sites in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. Strong associations exist within the 100kb region from DQB1 to DRB1, suggesting a low frequency of recombination in this region. Conversely, a lack of association between alleles of TAP1 and TAP2 (about 15kb) has been observed, suggesting that recombination occurs here with relative frequency. Analysis of familial-derived recombinant chromosomes is likely to enrich the current knowledge of recombination across the class II region which has been derived from disequilibrium studies of known class II haplotypes. Families containing recombinant chromosomes within the 700kb interval between the DPB1 and DRB1 genes have been identified for such purposes. Using single-strand conformation polymorphism analysis, 122 novel polymorphic markers distributed throughout the class II region were identified and used to delineate the site of crossover events in 26 class II recombinant chromosomes. The three regions where recombination occurred most frequently are as follows: the 45kb interval between DN-A and RING3 in 10 cases, the 40kb interval between DQB3 and DQB1 in 6 cases, and an 8.8kb segment of the TAP2 gene in 3 cases. The final seven recombinant chromosomes require further resolution, but all of these potentially fall within one of the three intervals mentioned. Two of the three TAP2 recombinant chromosomes have been mapped to overlapping 139bp and 850bp segments of intron 2. Associations between polymorphic markers immediately flanking each of the 3 defined regions using data from unrelated individuals will be compared with the familial recombination data observed in these regions.     

Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus

Recombinant foot-and-mouth disease viruses were isolated from cells infected with a mixture of temperature-sensitive (ts) mutants belonging to different subtype strains. In order to select for recombination events in many different regions of the genome, crosses were performed between various pairs of mutants, with ts mutations in different regions of the genome. ts+ progeny were analysed by electrofocusing virus-induced proteins and RNase T1 fingerprinting of their RNA. All but 5 out of 43 independent isolates, from nine crosses, proved to have recombinant RNA genomes. Maps of these genomes, based on a knowledge of the locations of the unique oligonucleotides, were constructed. Most could be interpreted as being the products of single genetic cross-overs, although three recombinants were formed by two cross-overs each. Cross-overs in at least twelve distinct regions of the genome were identified. This evidence of a large number of recombination sites suggests that RNA recombination in picornaviruses is a general, as opposed to a site-specific, phenomenon.     

Identification of a recombination event narrowing the Lafora disease gene region.

Patients affected with progressive myoclonus epilepsy of the Lafora type present during late adolescence with a characteristic EEG pattern and Lafora bodies seen on skin biopsy. The critical region for the Lafora gene has been localised to chromosome 6q24 flanked by the dinucleotide repeat markers D6S292 and D6S420. This study for linkage of markers from the candidate gene region was performed in a previously unpublished family affected with Lafora disease. EEG and skin biopsy evaluation for Lafora bodies were performed on five of eight family members followed for seizure activity. Haplotype and linkage analysis of DNA from five family members were carried out using the nine dinucleotide repeat markers reported in the common region of homozygosity by Serratosa et al in 1995. The present study of an additional family affected by Lafora disease has narrowed the 17 cM critical region for the Lafora disease gene on chromosome 6q24 to a 4 cM region flanked by markers D6S308 and D6S311.     

Emergence of a group 3 coronavirus through recombination

Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.     

Responses of mice to murine coronavirus immunization

Summary Oral and/or intranasal inoculation of susceptible mouse genotypes with the JHM strain of mouse hepatitis virus (MHV-JHM) consistently results in T cell dysfunction as reflected by in vitro proliferative responses to mitogens or allogeneic cells. One approach to examining the mechanism responsible for the observed functional T cell suppression is to determine whether virus replication is required for its induction. To this end, mice were inoculated oronasally with MHV-JHM that was inactivated with short-wave ultraviolet light, betapropiolactone or psoralen. Mice were also inoculated with live MHV-JHM after recovery from homotypic or heterotypic MHV infection. Spleen cells from BALB mice inoculated oronasally with inactivated MHV-JHM yielded extremely variable in vitro proliferative responses after concanavalin A stimulation. MHV-susceptible mice exposed oronasally or intraperitoneally to virus inactivated by any of the minimum effective treatments failed to seroconvert. Immunization with psoralen-treated virus intraperitoneally in Freund's complete adjuvant or oronasally failed to protect from live virus challenge, but survivors had elevated virus-specific serum IgG antibody titers compared to mock-immunized controls at two weeks post-challenge. Spleen cells from mice that were challenged after recovery from homotypic live virus infection did not exhibit the profound in vitro T cell suppression normally observed during the acute stage of primary infection. In contrast, MHV-JHM challenge of mice vaccinated with heterotypic live MHV-S resulted in significantly depressed in vitro T cell function. The combined data suggest that either virus replication or exposure to more concentrated antigen may be required for induction of the dramatic T cell dysfunction that occurs as a consequence of MHV-JHM infection as well as for a detectable MHV-specific humoral response.     

Identification of putative polymerase gene product in cells infected with murine coronavirus A59

The virion RNA of mouse hepatitis virus, strain A59 (MHV-A59) is believed to be the mRNA for the viral RNA-dependent RNA polymerase. The cell-free translation of virion RNA results in the synthesis of two predominant products p220 and p28 (M. R. Denison and S. Perlman, 1986, J. Virol. 60, 12-18). p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. When infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells.