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LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60gag). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus complex that contains causative defective, and ecotropic helper, retroviruses. We previously demonstrated that the MAIDS-resistant H-2(d) strains BALB/cByJ and C57BL/KsJ generate MHC class I (K(d)) restricted virus-specific CD8(+) cytolytic T lymphocytes (CTLs) that lyse cells expressing either defective or ecotropic gag proteins. In contrast, the congenic BALB.B and closely related C57BL/6J MAIDS-susceptible H-2(b) strains were unable to serve as a source of gag-specific CTLs (Schwarz and Green, 1994), suggesting that anti-gag CTLs might provide a basis for resistance to MAIDS. Although its susceptibility to MAIDS was unknown, the (BALB/c x C57BL/6J) F(1) (CBY6F(1)) strain could also produce H-2(d)-, but not H-2(b)-, restricted, anti-gag CTLs (Schwarz and Green, 1994). Because of this correlation between anti-gag CTLs and resistance to MAIDS, it was important to provide more direct evidence in support of CTL-mediated protection and to determine both the fine specificity of CByB6F(1) anti-gag CTLs, in comparison with the resistant C57BL/Ks and BALB/c strains, and the susceptibility of this F(1) strain to LP-BM5-induced MAIDS. We report here that no symptoms of MAIDS were observed in CBY6F(1) (H-2(dxb)) mice. For F(2) mice, in contrast to the high susceptibility of H-2(b/b) mice, 77% of H-2(d/d) and 81% of H-2(b/d) F(2) mice did not exhibit MAIDS after LP-BM5 infection. These results are in contrast to other published studies that concluded that susceptibility, rather than resistance, is dominant in F(1) (resistant x susceptible or susceptible x resistant) mice. We also show that CBY6F(1) anti-gag CTLs exhibit a fine specificity shared by the MAIDS-resistant BALB/c and C57BL/Ks strains, that is, the immunodominant gag epitope, SYNTGRFPPL, encoded by an alternative open reading frame. Together with our direct demonstration here that in vivo monoclonal antibody (mAb) depletion of CD8(+) T cells converts genetically resistant mice to MAIDS susceptibility, these data on the ability to mount anti-ORF2/SYNTGRFPPL, gag-specific CTL responses strongly suggest that CTLs are a primary factor in determining MAIDS resistance. Accordingly, given the K(d)-restricted nature of the CTLs, the main genetic determinant of resistance appeared to be the codominant expression of the resistant H-2(d) haplotype. Interestingly, however, 19% of H-2(d/b) and 23% of the H-2(d/d) F(2) mice had at least one clinical aspect of MAIDS, suggesting that a non-MHC genetic determinant(s) can negatively influence T-cell protection and thus disease outcome, (Copyright 2000 Academic Press.)

The murine AIDS (MAIDS) virus has a unique sequence in its gag p12 region. A transcript which hybridizes with this sequence is expressed in normal C57BL/6 mice. This transcript has been proposed to be the origin of the MAIDS virus, since the virus was originally isolated from radiation-induced leukemic C57BL/6 mice. The transcript, designated Edv, was molecularly cloned and sequenced. Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic defective MAIDS virus has 16-bp deletions and a 1-bp insertion in the 5' and 3' regions of the gag p12 sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the gag p12 region of the MAIDS virus is less homologous to that of the helper virus and Edv transcript due to the frameshift mutations. This suggested that the MAIDS virus was generated by such frameshift mutations in the gag p12 region during recombination between the helper virus and the Edv or a related sequence.

The mechanisms responsible for development of profound immunodeficiency and extensive lymphoproliferation that characterize infection of different species with retroviruses are only partially understood. In mice, it has been shown the activities of an unusual Gag protein are necessary and sufficient to induce these abnormalities in a syndrome designated mouse AIDS (MAIDS). Current studies suggest that complex, antigen-driven interactions between T cells and B cells result in polyclonal activation of both types of lymphocytes, aberrant cytokine production and late lymphomas.

A defective murine leukemia virus is the causative agent of murine acquired immunodeficiency syndrome (MAIDS). We have cloned cDNAs from both virus infected and non-infected cells using the PCR methods with primers corresponding to the franking sequence of the unique p12 gag gene. Sequence analysis of these cDNA clones revealed: (i) the presence of endogenous virus related to MAIDS virus in C57BL/6 mice, (ii) B cell lineage specific expression of endogenous virus and (iii) extensive heterogeneity of MAIDS virus recovered from virus infected cells due to the recombination of the related viruses (defective pathogenic virus, ecotropic virus and endogenous virus). These findings suggest that the creation of virus variants in infected cells may play an important role in virus pathogenesis and escape from immune attack during the development of MAIDS.

Blood cytopenia is a common feature in HIV infection, occurring in up to 70% of patients with AIDS. Since at present it is not clear to what extent this is intrinsic to HIV infection or due to opportunistic infections and antiretroviral agents we have investigated the long-term effects of conventional and new antiviral drugs on the bone marrow of normal and immunodeficient mice. The results show that azidothymidine (AZT), dideoxycytidine (DDC) and dideoxycytidine 5'-triphosphate (DDCTP) alone or in combination are all effective in inhibiting the expression of the retroviral protein Pr60gag in bone marrow cells. However, DDCTP was the most effective in preventing bone marrow cytopenia. Combined treatments of AZT plus DDCTP result in a reduction in erythroid precursors compared to that resulting from DDCTP administration, while DDC plus DDCTP results in a differential cell count similar to that found in uninfected mice. Thus, the bone marrow in murine AIDS may prove useful as a model for therapy of retroviral infections and for treating blood cytopenias.

Pr60gag appears to be the only protein encoded by the murine AIDS (MAIDS)-defective virus. To study the role of Pr60gag or some other sequences of the viral genome in the pathogenicity of the virus, we have generated mutants of the defective viral genome. These mutant defective viruses, prepared as helper-free stocks, were inoculated into susceptible C57BL/6 mice. Mutant Du5H-A virus, which had a stop codon within gag MA(p15), did not induce target cell proliferation or MAIDS. Mutants Du5H-B and -C encoded truncated Pr60gag proteins containing, respectively, MA(p15)-p12 or MA(p15)-p12 and part of CA(p30). These mutants showed a very limited capacity to induce early cell expansion and were poorly pathogenic. Only recombinant (revertant) viruses were recovered from organs of diseased mice inoculated with these two mutants. Mutant Du5H-D was generated by deleting 1.4 kbp of the 3'-end sequences, outside the gag coding region. The levels of RNA and proteins made by this mutant were low. This mutant also reverting frequently but was nevertheless able to induce MAIDS at a low efficiency without reverting. Our results indicate that the Pr60gag protein is necessary and sufficient to induce MAIDS. These data also suggest that the Pr60gag protein needs to be relatively intact to be fully pathogenic. In addition, our study shows a very high reversion rate of some mutants and emphasizes the need to check for the presence of revertant (recombinant) viruses in diseased organs when working with mutants of the MAIDS-defective virus.