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Mouse hepatitis virus has been shown to undergo RNA recombination at high frequency during mixed infection. Temperature-sensitive mutants were isolated using 5-fluorouracil and 5-azacytidine as mutagen. Six RNA+ mutants that reside within a single complementation group mapping within the S glycoprotein gene of MHV-A59 were isolated which did not cause syncytium at the restrictive temperature. Using standard genetic techniques, a recombination map was established that indicated that these mutants mapped into two distinct domains designated F1 and F2. These genetic domains may correspond to mutations mapping within the S1 and S2 glycoproteins, respectively, and suggest that both the S1 and S2 domains are important in eliciting the fusogenic activity of the S glycoprotein gene. In addition, assuming that most distal ts alleles map roughly 4.0 kb apart, a recombination frequency of 1% per 575-676 bp was predicted through the S glycoprotein gene. Interestingly, this represents a threefold increase in the recombination frequency as compared to rates predicted through the polymerase region. The increase in the recombination rate was probably not due to recombination events resulting in large deletions or insertions (greater than 50 bp), but rather was probably due to a combination of homologous and nonhomologous recombination. A variety of explanations could account for the increased rates of recombination in the S gene.

MHV-A59 temperature-sensitive mutants, representing one RNA+ and five RNA− complementation groups, were isolated and characterized by genetic recombination techniques. Maximum recombination frequencies occurred under multiplicities of infection greater than 10 each in which 99.99% of the cells were co-infected. Recombination frequencies between different is mutants increased steadily during infection and peaked late in the virus growth cycle. These data suggest that recombination is a late event in the virus replication cycle. Recombination frequencies were also found to range from 63 to 20,000 times higher than the sum of the spontaneous reversion frequencies of each is mutant used in the cross. Utilizing standard genetic recombination techniques, the five RNA− complementation groups of MHV-A59 were arranged into an additive, linear, genetic map located at the 5′ end of the genome in the 23-kb polymerase region. These data indicate that at least five distinct functions are encoded in the MHV polymerase region which function in virus transcription. Moreover, using well-characterized is mutants the recombination frequency for the entire 32-kb MHV genome was found to approach 25% or more. This is the highest recombination frequency described for a nonsegmented, linear, plus-polarity RNA virus.

Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNγ+T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.

The p12I protein, encoded by the pX open reading frame I of the human T-lymphotropic virus type 1 (HTLV-1), is a hydrophobic protein that localizes to the endoplasmic reticulum and the Golgi. Although p12I contains 4 minimal proline-rich, src homology 3–binding motifs (PXXP), a characteristic commonly found in proteins involved in signaling pathways, it has not been known whether p12I has a role in modulating intracellular signaling pathways. This study demonstrated that p12I binds to the cytoplasmic domain of the interleukin-2 receptor (IL-2R) β chain that is involved in the recruitment of the Jak1 and Jak3 kinases. As a result of this interaction, p12I increases signal transducers and activators of transcription 5 (STAT5) DNA binding and transcriptional activity and this effect depends on the presence of both IL-2R β and γc chains and Jak3. Transduction of primary human peripheral blood mononuclear cells (PBMCs) with a human immunodeficiency virus type 1–based retroviral vector expressing p12I also resulted in increased STAT5 phosphorylation and DNA binding. However, p12I could increase proliferation of human PBMCs only after stimulation of T-cell receptors by treatment of cells with low concentrations of αCD3 and αCD28 antibodies. In addition, the proliferative advantage of p12I-transduced PBMCs was evident mainly at low concentrations of IL-2. Together, these data indicate that p12I may confer a proliferative advantage on HTLV-1–infected cells in the presence of suboptimal antigen stimulation and that this event may account for the clonal proliferation of infected T cells in vivo.

Coronaviruses are common respiratory and gastrointestinal pathogens of mammals and birds. Not only do they cause about 15–20% of the common colds in humans, they are also occasionally associated with infections of the lower respiratory tract and central nervous system1. The prototype, mouse hepatitis virus (MHV), contains a 32 kb genomic RNA which encodes two large orfs at the 5′ end, designated orf la and orf lb. Orf lb contains highly conserved polymerase, helicase and metal binding motifs typical of viral RNA polymerases while orf 1 a contains membrane and cysteine rich domains, and serine-and poliovirus 3c-like protease motifs1. The large size of the genome coupled with it’s unique replication strategy and high recombination frequencies during mixed infection predict a considerable capacity to evolve1,2,3.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.

Mouse hepatitis virus (MHV), a member of the Coronaviridae, contains a single-stranded, nonsegmented, plus-polarity RNA of 8.0 × 106 daltons molecular weight1 . The ~32KB genomic RNA is organized into seven genetic regions each encoding one or more viral proteins 1,2. In the virion, the RNA is enclosed in a helical nucleocapsid structure constructed from multiple copies of a 50-60KD phosphorylated nucleocapsid protein(N). The viral envelope is derived from modified host internal membranes and contains two virus-encoded glycoproteins designated M (gp23) and S(gpl80/90) 1,3.