Defective Murine Leukemia Virus Research Articles

A new one-step RT-PCR method for virus quantitation in murine AIDS

The causative agent of murine AIDS (MAIDS) in C57BL/6 mice, is a defective murine leukemia virus (BM5d) that requires the replication-competent helper virus (BM5e). Since this animal model of immunodeficiency, which shows many similarities to human AIDS, is also used to test the efficacy and toxicity of antiretroviral drugs, a method that allows the quantitative detection of both viruses would be very useful also if hampered potentially by endogenous viral sequences usually present in mice. While BM5d alone could induce the disease, the effect of BM5e on the immune system of diseased mice is unclear. A specific and reliable one-step RT-PCR method was developed for the co-amplification, with the same efficiency, of BM5d or BM5e with ß-actin used as an internal standard. The standard curves produced with cloned cDNA sequences (ß-actin and BM5d or BM5e) assure that all samples are analyzed during the exponential phase of the reaction. Using this new assay which provided a dynamic range of at least four-log-unit, the ratio of initial absolute amounts of the virus and ß-actin RNA was determined, obtaining quantitative information on virus-specific cellular-transcript in the lymph nodes and spleen during the natural history of the disease and during therapeutic regimens.

Activation of the ubiquitin proteolytic system in murine acquired immunodeficiency syndrome affects IkappaBalpha turnover.

Murine acquired immunodeficiency syndrome (MAIDS) is a complex immunopathology caused by a defective murine leukemia virus (LP-BM5) that mainly targets B-lymphocytes. Lymphadenophathy, splenomegaly, hypergammaglobulinemia and progressive immunodeficiency are prominent features of MAIDS. Previously, we showed that the ubiquitin proteolytic system was upregulated in infected lymph nodes [Crinelli, R., Fraternale, A., Casabianca, A. & Magnani, M. (1997) Eur. J. Biochem. 247, 91-97]. In this report, we demonstrate that increased 26S proteasome activity is responsible for accelerated turnover of the IkappaBalpha inhibitor in lymph node extracts derived from animals with MAIDS. The molecular mechanisms mediating IkappaBalpha proteolysis involved constitutive phosphorylation of IkappaBalpha at Ser32 and Ser36 and subsequent ubiquitination, suggesting persistent activation of an NF-kappaB inducing pathway. Interestingly, enhanced IkappaBalpha degradation did not result in enhanced NF-kappaB DNA binding activity, but rather in a different subunit composition. The modulation of NF-kappaB/IkappaB system may affect multiple immunoregulatory pathways and may in part explain the mechanisms leading to the profound immune dysregulation involved in MAIDS pathogenesis.

CD40-deficient mice infected with the defective murine leukemia virus LP-BM5def do not develop murine AIDS but produce IgE and IgG1 in vivo.

CD40-deficient mice, when inoculated with the LP-BM5def murine retorvirus, become infected and show virus expression similar to wild-type mice. However, unlike the wild-type mice, CD40-deficient mice do not develop symptoms of immunodeficiency, lymphoproliferative disease and the typical histological changes in the lymphoid tissue. These results show that the CD40-CD40 ligand (CD40L) interaction in vivo is essential for anergy induction and the subsequent development of immunodeficiency and pathologic expansion of lymphocytes. Infected CD40-deficient mice and their littermates express a similar pattern of cytokine mRNA, which is not biased towards a Th2 phenotype. Nevertheless, hypergammaglobulinemia is induced in infected wild-type and CD40-deficient mice. Surprisingly, murine AIDS infection even induces IgE production in CD40-deficient mice in vivo. Our data demonstrate that antibody class switch to IgE and IgG1 can be induced by a retroviral infection in vivo even in the absence of CD40-CD40L interaction and an apparent switch to a Th2 cytokine production.

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.

Involvement of temperature sensitive syncytium inducing VSV or defective retroviruses in the development of spongiform encephalopathies

Various enveloped viruses can induce syncytia in competent cells. Some temperature-sensitive mutants can express the trans-membrane viral G antigen under non-permissive conditions. The G antigen can then migrate at long distances, engulfing thousands of cells without producing any virus. When a temperature-sensitive vesicular stomatitis virus (VSV) infects a sensitive host, and under the condition that the G antigen is expressed, spongiosis can be induced in the central nervous system in the absence of detectable virus multiplication. We postulate that such a mechanism might be observed with various enveloped viruses, as recently illustrated with knock-out mice experimentally infected with defective murine leukemia virus (MULV).

Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus.

The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.

Suppression of Hematopoietic Support Function Is Associated with Overexpression of lnterleukin-4 and Transforming Growth Factor-β1 in LP-BM5 Murine-Leukemia-Virus-lnfected Stromal Cell Lines

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus (MuLV) is a disease with many similarities to human AIDS. Our previous studies demonstrated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures could be attributed to a defective hematopoietic stroma. We report now the generation of permanent stroma cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by the polymerase chain reaction for detecting viral genome expression of the p12 envelope glycoprotein. The ability of these cell lines to support in vitro hematopoiesis was evaluated. The results demonstrated that when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic progenitors, whereas in virus-infected progenitors was suppressed. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for interleukin (IL)-1 beta, IL-2, IL-3, IL-6, IL-7, IL-10, interferon, tumor necrosis factor-alpha and stem cell factor. However, infection was associated with higher expression of IL-4 and transforming growth factor-beta 1. These findings demonstrate that infected stomal cell lines generate a defective hematopoietic microenvironment to produce altered cytokine expression and faulty hematopoiesis. Further characterization of these defective cell lines should assist elucidation of the mechanism(s) whereby retroviruses alter hematopoiesis ultimately leading to the generation of immunodeficiency.

Apoptotic death of lymphocytes in murine acquired immunodeficiency syndrome: Involvement of Fas‐Fas ligand interaction

Murine acquired immunodeficiency syndrome (MAIDS) is caused by a defective murine leukemia virus. The disease is characterized by abnormal lymphoproliferation, impaired T and B cell function and aberrant regulation of cytokines. Both T and B lymphocytes show activated phenotypes, but undergo apoptotic death with characteristic DNA fragmentation. These results indicate the presence of a continuous activation death pathway of the lymphocytes in MAIDS. Overexpression of the bcl-2 transgene in lymphocytes showed no effect on the apoptotic cell death or on the development of the disease. In contrast, mice carrying mutations in either Fas or Fas ligand exhibited accelerated progression of the disease upon infection with MAIDS virus. These results suggest the involvement of Fas-Fas ligand system in the pathogenesis of MAIDS.

Sequence heterogeneity of murine acquired immunodeficiency syndrome virus: the role of endogenous virus.

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.

Unbiased usage of T‐cell receptor β variable region genes in peripheral blood cells of hepatitis C patients: No correlation with superantigen effect

Hepatitis C virus (HCV) infection frequently causes chronic hepatitis and lack of virus clearance in these patients. In addition, many patients infected by HCV also present with hypergammaglobulinemia in the early stage of chronic infection. These observations raise a possible viral superantigen effect induced by HCV, because viral superantigen found in human immunodeficiency virus (HIV) or in replication of defective murine leukemia virus (MuLV) is associated with T-cell dysfunction and polyclonal activation of B cells. The possibility was investigated of whether HCV encodes any superantigen by analyzing the usage of T-cell receptor (TCR) from the peripheral blood lymphocytes (PBL) of patients with chronic hepatitis C. Two groups, one with hypergammaglobulinemia and the other without hypergammaglobulinemia, were studied for the usage of TCR beta chain by reverse transcription-polymerase chain reaction (RT-PCR) analysis. It was found that all genes of V beta variable chain were used in the PBL of these patients. Furthermore, there was no significant difference of the TCR expression pattern between these two groups, nor a complete deletion of a particular T-cell subset in either group. These results do not provide evidence for HCV superantigen, but indicate that the TCR usage in the patients was neither defective nor biased.

Myristylation of Pr60gag of the murine AIDS-defective virus is required to induce disease and notably for the expansion of its target cells.

Murine AIDS (MAIDS) is characterized by severe lymphadenopathy and splenomegaly. The proliferation of the infected target B cells is also an important manifestation of the disease (M. Huang, C. Simard, D. G. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). The etiologic agent of MAIDS is a defective murine leukemia virus that is deleted of most of its pol and env genes and appears to encode a single protein, the Gag precursor Pr60gag protein. Pr60gag is myristylated and attached to the plasma membrane. To study the role myristylation on the function of Pr60gag, we have generated a myristylation-negative (Myr-) mutant of the MAIDS defective virus. We found that Myr- Pr60gag interacted less tightly with the plasma membrane. In addition, the Myr- MAIDS defective virus mutant was unable to induce expansion of infected cells and was nonpathogenic. These results emphasize the essential role of Pr60gag in the disease process. Our data also suggest that Pr60gag, once recruited to the cell membrane through its myristylation, interacts with other membrane-bound effectors to send signals to induce proliferation of the infected cells and to initiate immune dysfunctions.

Stromal cell lines derived from LP-BM5 murine leukemia virus-infected long-term bone marrow cultures impair hematopoiesis in vitro

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

Characteristics of CD4+ T Cells Which Transfer Murine AIDS (MAIDS)

The murine acquired immunodeficiency syndrome (MAIDS) is caused in susceptible C57BL/6 (B6) mice by a defective murine leukemia virus (MuLV) and resembles human AIDS in several respects. The disease is characterized by hypergammaglobulinemia, polyclonal B cell activation, lymphadenopathy, and generalized immunosuppression within 5-8 weeks postinfection. The virus has been shown to infect B cells and macrophages and both T and B cells are required for MAIDS development. The manner in which T cells contribute to the disease process is got known. We report here that this retroviral infection leads to induction of a Thy-CD4+ T cell subpopulation capable of transferring all the symptoms of MAIDS disease to normal B6 and B6 nu/nu. Essentially 100% or T cells recovered from B6 nu/nu mice, injected with CD4+ T cells from B6 MAIDS animal, is of the Thy CD4+ phenotype. The proliferation of these T cells in culture and their ability to cause MAIDS in SCID mice is totally dependent on the presence of B cells. These T cells do not exhibit significant Vβ restriction of their T cell receptors (TCR) and, by PCR analysis, have defective virus-specific sequences in the cellular genome. By several criteria, however, these cells do not produce the infectious virus. These results suggest that a B-cell-dependent population of CD4+ T cells from MAIDS animals, in the absence of detectable infectious virus production, has the ability to transfer MAIDS-like disease.

An IFN-gamma-dependent pathway plays a critical role in the pathogenesis of murine immunodeficiency syndrome induced by LP-BM5 murine leukemia virus.

The murine acquired immunodeficiency syndrome (MAIDS) caused by a defective murine leukemia virus produces severe immunodeficiency with abnormal lymphoproliferation and hypergammaglobulinemia. The presence of both CD4+ T cells and B cells is critical for the development of this disease. Remarkably elevated mRNA expression for IFN-gamma and IL-10 was observed in spleen cells of C57BL/6 mice starting from the early phase of viral infection. IFN-gamma production was induced by spleen cells from virus-infected mice upon stimulation with concanavalin A or lipopolysaccharide in both the early and late phases of MAIDS progression. When mice that had been passively administered anti-IFN-gamma mAb were infected with the virus, the development and progression of lymphadenopathy, immunodeficiency and elevated levels of serum IgG2a associated with MAIDS were delayed. Treatment with anti-IL-4 or anti-IL-10 mAb in place of anti-IFN-gamma mAb did not induce the delayed progression of MAIDS. These data support the concept that IFN-gamma-dependent pathway may be involved in the development of MAIDS.

Resistance of mice deficient in IL-4 to retrovirus-induced immunodeficiency syndrome (MAIDS)

The murine acquired immunodeficiency syndrome (MAIDS) is induced by a defective murine leukemia virus and has many symptoms similar to those found in patients infected with the human immunodeficiency virus. The presence of both B cells and CD4+ T cells is critical for the development of the disease. Furthermore, a Th2 cytokine response dominates during the progression of the disease. When interleukin-4 (IL-4)-deficient mice that are defective in Th2 cytokine responses were infected, there was no lethality, and the development of the T cell abnormalities associated with MAIDS was delayed. These data suggest that IL-4 or a Th2 response is involved in the development of retrovirus-induced immunodeficiency in mice.

Effect of lithium in murine immunodeficiency virus infected animals.

Murine AIDS (MAIDS) is a disease that shows many similarities to human HIV infection. The etiological agent of MAIDS is a defective murine leukemia virus that seems to be able to induce disease in the absence of viral replication. This animal model has been useful in stimulating the search of answers to questions and the formation of new hypotheses related to human AIDS. The monovalent cation lithium can influence a number of immunohematopoietic cell types and cellular processes where proliferation and differentiation occur. We describe here the result of in vivo studies investigating the effect of lithium treatment on MAIDS-infected mice. Viral control and lithium-treated animals were monitored for survival and development of MAIDS pathology. MAIDS animals treated with lithium demonstrated a marked reduction in their development of lymphadenopathy and splenomegaly. Both MAIDS control and lithium-treated virus-infected mice developed evidence of lymphoma; however, the involvement was much more massive both at the gross and microscopic levels in the MAIDS control compared with the lithium-treated mice. These data suggest that lithium may be effective in modulating murine immunodeficiency virus infection and raise important questions related to the potential role lithium may play in the pathophysiological processes associated with retroviral infections.

Susceptibility of inbred strains of mice to murine AIDS (MAIDS) correlates with target cell expansion and high expression of defective MAIDS virus.

Murine AIDS (MAIDS) is readily induced by the Duplan strain of defective murine leukemia virus in susceptible C57BL/6 mice. To identify mouse strains resistant to MAIDS, and to understand the genetic factors controlling susceptibility to the disease, we screened more than 20 inbred strains of mice for their susceptibility to MAIDS. For this study, mice of the Fv-1n/n, Fv-1b/b, or Fv-1n/b genotype were inoculated with stocks of defective MAIDS virus pseudotyped with N-tropic, B-tropic, or NB-tropic helper murine leukemia virus, respectively. Strains could be classified as susceptible, resistant, or moderately resistant. None of the individual H-2 haplotypes examined appears to explain resistance to MAIDS by itself. However, a very good correlation between the susceptibility or resistance phenotype and the presence or absence of defective proviral DNA and RNA in the spleen of these animals was found. Since the presence of defective proviral DNA and RNA reflects the oligoclonal proliferation of the cells infected by the defective MAIDS virus, our results strongly suggest that this target cell expansion is genetically controlled and is necessary and perhaps even sufficient for the development of the disease.

Murine acquired immunodeficiency syndrome (MAIDS): an animal model to study the AIDS pathogenesis

Murine AIDS (MAIDS) is a disease that shows many similarities with human AIDS. Several immunological parameters of the disease have been analyzed and genetic studies have mapped a gene (or genes) of resistance in the H-2 complex and shown that the genetic background of the mouse can significantly modify some features of the disease. The etiologic agent of MAIDS is a defective murine leukemia virus that seems able to induce disease in the absence of virus replication. This defective virus induces proliferation of its target cells and the cell expansion was found to be oligoclonal, thus suggesting that the immunodeficiency observed in these mice is a paraneoplastic syndrome. The excellent response of MAIDS mice to antineoplastic agents is consistent with this notion. This animal model has already been useful in stimulating the emergence of novel questions and the formulation of new hypotheses about human AIDS, namely about the role of defective HIV, the role of HIV replication in the progression of the disease, and the importance to identify the target cells of HIV in vivo. Although MAIDS and AIDS are not identical and are induced by retroviruses of different classes, the availability of such a model in an easily accessible small animal species, whose genetics is very sophisticated, may be instrumental in understanding the pathogenesis of AIDS if some of the cellular and molecular affected pathways are common in both diseases.

The effect if cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines

The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65 gag in a defective murine leukemia virus (334C) producing cell lines (MJD-54, clone 2) the amount of the Pr65 gag remaining producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65 gag remaining in the presence of cerulenin (20 μg/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65 gag polyprotein, since the env precursor polyprotein Pr80 env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells.

Comparison of murine sarcoma viruses in nonproducer and S + L - -transformed cells.

The biologic properties of murine sarcoma viruses in nonproducer and S(+)L(-) cells were compared. Each virus was found to be genetically stable through at least two cycles of rescue and transmission to new cells. There was no evidence for production of a defective murine leukemia virus by S(+)L(-) cells. The results are most consistent with the hypothesis that the sarcoma genome in S(+)L(-) cells contains information for replication functions which the virus in nonproducer cells either lacks or does not express.