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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.