Murine AIDS Virus Research Articles

면역결핍 모델에서 β-1,3/1,6-glucan과 유산균을 이용한 in vivo 면역 활성 조절 효과

In this study, β-1,3/1,6-glucan, lactic acid bacteria, and β-1,3/1,6-glucan+lactic acid bacteria were tested for 10 weeks using an immunodeficient animal model infected with LP-BM5 murine AIDS virus On the immune activity. Cytokines production, plasma immunoglobulin concentration, T cell and B cell proliferation were measured. As a result, the T cell proliferative capacity which was weakened by immunization with LP-BM5 murine AIDS virus increased significantly T cell proliferative capacity compared with the red ginseng control group. B cell proliferative capacity was significantly higher than the infected control group. Increased B cell proliferation was reduced. In the cytokine production, IL-2, IL-12 and IL-15 in the Th1-type cytokine increased the secretion of IL-2, IL-12 and IL-15 compared to the infected control. The proliferative capacity of the treated group was higher than that of the mixed treatment group. TNF-α was significantly decreased compared with the infected control group. The IL-4, IL-6 and IL-10 levels were significantly inhibited in the infected control group and the Th1/Th2 type cytokine expression was regulated by immunohistochemistry. IgE, IgA, and IgG levels were significantly lower in the immunoglobulin secretion assay than in the control. As a result, the immunomodulatory effect of β-1,3/1,6-glucan+lactic acid bacteria was confirmed by mixing with LP-BM5 murine AIDS virus-infected immunodeficient animal model.

Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex

Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from healthy human semen have been shown to inhibit HIV-1 replication and to impair progeny virus infectivity. In this study, we examined the ability of healthy human semen exosomes to restrict HIV-1 and LP-BM5 murine AIDS virus transmission in three different model systems. We show that vaginal cells internalize exosomes with concomitant transfer of functional mRNA. Semen exosomes blocked the spread of HIV-1 from vaginal epithelial cells to target cells in our cell-to-cell infection model and suppressed transmission of HIV-1 across the vaginal epithelial barrier in our trans-well model. Our in vivo model shows that human semen exosomes restrict intravaginal transmission and propagation of murine AIDS virus. Our study highlights an antiretroviral role for semen exosomes that may be harnessed for the development of novel therapeutic strategies to combat HIV-1 transmission.

Human semen contains exosomes with potent anti-HIV-1 activity.

BackgroundExosomes are membranous nanovesicles secreted into the extracellular milieu by diverse cell types. Exosomes facilitate intercellular communication, modulate cellular pheno/genotype, and regulate microbial pathogenesis. Although human semen contains exosomes, their role in regulating infection with viruses that are sexually transmitted remains unknown. In this study, we used semen exosomes purified from healthy human donors to evaluate the role of exosomes on the infectivity of different strains of HIV-1 in a variety of cell lines.ResultsWe show that human semen contains a heterologous population of exosomes, enriched in mRNA encoding tetraspanin exosomal markers and various antiviral factors. Semen exosomes are internalized by recipient cells and upon internalization, inhibit replication of a broad array of HIV-1 strains. Remarkably, the anti-HIV-1 activity of semen exosomes is specific to retroviruses because semen exosomes blocked replication of the murine AIDS (mAIDS) virus complex (LP-BM5). However, exosomes from blood had no effect on HIV-1 or LP-BM5 replication. Additionally, semen and blood exosomes had no effect on replication of herpes simplex virus; types 1 and 2 (HSV1 and HSV2). Mechanistic studies indicate that semen exosomes exert a post-entry block on HIV-1 replication by orchestrating deleterious effects on particle-associated reverse transcriptase activity and infectivity.ConclusionsThese illuminating findings i) improve our knowledge of the cargo of semen exosomes, ii) reveal that semen exosomes possess anti-retroviral activity, and iii) suggest that semen exosome-mediated inhibition of HIV-1 replication may provide novel opportunities for the development of new therapeutics for HIV-1.

Loss of Ubr1 Cooperatively Synergizes with HOX11 in B Cell Lymphomagenesis.

The HOX11 homeobox gene was originally identified at the recurrent t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5–7% of T cell acute lymphoblastic leukemias (T-ALLs). Transgenic mice ectopically expressing HOX11 in the B cell compartment die in their second year of life due to the onset of mature B cell lymphomas. However, the long latency prior to the development of leukemia has led to the hypothesis that additional mutations are necessary prior to the onset of full-blown malignancy. To identify collaborating genetic loci responsible for HOX11-induced B cell lymphomagenesis, proviral insertional mutagenesis, using the mature B cell-specific retrovirus, the murine AIDS (mAIDS) virus, was used. In eight of ten animals, there was an acceleration of development of B cell lymphomas, manifested by the development of a mediastinal mass comprising predominantly of mature IgM+ IgD+ B cells. Histological analysis revealed expansion of splenic germinal centres and hyperplasia of adjacent lymph nodes, consistent with diffuse large B cell lymphoma. Using the provirus as a molecular tag, we identified Ubr1 as a frequent site of proviral insertion. Three mice exhibited an insertion into the 10th exon of the Ubr1 gene, with two animals exhibiting an identical insertion at nucleotide 1295 and another animal exhibiting an insertion at nucleotide 1251. Insertion into this genomic region was confirmed by Southern blotting demonstrating the presence of a rearranged Ubr1 allele, and by the ability to generate a PCR amplicon across the viral-genome junction. Western immunoblot analysis revealed down-regulated expression of the Ubr1 gene product subsequent to viral integration. Ubr1 is a member of the E3 ubiquitin ligase family and participates in the ubiquitin-dependent proteolytic pathway. Among its numerous targets, Ubr1 controls the timely degradation of cohesin subunits during mitosis. Consequently, Ubr1−/−S. cerevisiae are prone to chromosome loss due to chromosome malsegregation during anaphase. We sought to investigate possible similar effects in primary B lymphocyte cultures derived from HOX11Tg/Ubr1+/+ and HOX11Tg/Ubr1−/− mice, and to determine whether HOX11 overexpression in such a Ubr1-null background possesses any synergizing effects on the ploidy of these cells. Direct counting of chromosome numbers from chromosome spreads prepared from HOX11Tg/Ubr1−/− primary B lymphocytes cultured in vitro for 4–5 days revealed increased incidences of aneuploidy and chromosome loss relative to HOX11wt/Ubr1−/− controls (2n=39.51 vs. 2n=39.98). Similarly, micronucleus assays indicated increased presence of micronuclei in HOX11Tg/Ubr1−/− primary B lymphocyte cultures (5.2% vs 0.8%). Additionally, HOX11Tg B lymphocytes exhibit increased cyclin B1 expression and an ability to bypass G2-M arrest induced by the tyrosine kinase inhibitor, genistein. Therefore, the effect of HOX11 in exacerbating chromosome loss in these cultures may be associated with its ability to allow cells to bypass the G2-M cell cycle checkpoint, permitting the accrual of additional chromosome losses and cytogenic abnormalities en route to malignancy.

Induction of Murine AIDS Virus-Related Sequences after Burn Injury

To better understand the molecular signaling events leading to systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF), changes in gene expression profiles after burn injury were investigated by differential display. C57BLKS/J mice were subjected to 18% total body surface area (TBSA) full-thickness burn and various tissues were harvested at multiple time points after injury. Initial differential display revealed that retroviral transcripts similar to the envelope sequence of murine AIDS (MAIDS) virus were rapidly and transiently up-regulated after injury. Subsequent RT-PCR and DNA sequencing analyses confirmed the transient up-regulation of retroviral sequences similar to those of the MAIDS virus. In addition, the presence and induction of the subgenomic envelope transcripts of these MAIDS virus-related sequences, including a novel double spliced message, were identified after burn injury. These data suggest that the transcriptional efficiency of the integrated retroviral DNA and reactivation of defective MAIDS virus-related sequences may be affected by pathophysiological signals, such as burn injury. The elevated expression of these MAIDS virus-related retroviral sequences may affect the transcriptional activities of the flanking genes at the integration sites and may be a cause of altered local and systemic immune responses to burn-related stress.

Fish oil suppressed cytokines and nuclear factor-kappaB induced by murine AIDS virus infection

The effects of dietary fish oil on pro-inflammatory cytokines, nuclear factor-kappaB (NF-κB) and viral replication were studied in a murine model of AIDS. Sixty-four female C57BL/6 mice were divided into two dietary groups and fed either a corn oil or fish oil diet. After 4 weeks, each group was divided into two subgroups. One subgroup from each group was infected with LP-BM5 murine leukemia virus. At 4 and 10 weeks post-infection, one-half of the mice were sacrificed. Blood samples, and the spleens and livers were collected. The results demonstrated that when compared to corn oil, fish oil partially suppressed the virus-induced overproduction of serum IgG and IgM and the in vitro LPS-stimulated production of leukotriene B 4, tumor necrosis factor-α and interleukin-1β in splenocytes. Fish oil also partially suppressed NF-κB activation and viral replication in both tissues when compared to corn oil.

Induction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock.

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.

Delayed progression of murine AIDS in [formula omitted] mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene

C57BL 6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In constrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8 + T cells.

Heterosexual transmission of a murine AIDS virus.

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.

The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl.

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells.

Apoptosis by CD95 (Fas)-dependent and -independent mechanisms in Peyer's patch lymphocytes in murine retrovirus-induced immunodeficiency syndrome.

CD95 (Fas)/CD95 ligand (CD95 L)-mediated apoptosis is thought to be involved in the delayed progression of murine AIDS (MAIDS) induced by LP-BM5 murine leukemia virus (MuLV). We show evidence of apoptosis in lymphocytes of Peyer's patches (PP) at the early stage of MAIDS. Both T and B cells in PP expressed CD95 at the early stage of MAIDS and decreased in number thereafter. The decrease in T cells was not evident in CD95-mutated lpr mice with MAIDS, suggesting that CD95/CD95 L interaction is involved in the apoptosis of T cells in PP during the course of MAIDS. On the other hand, the number of B cells was also decreased in PP of lpr mice with MAIDS. The proliferative ability of B cells in PP of MAIDS mice in response to immunoglobulin M cross-linking or lipopolysaccharide was severely impaired, while the B cells normally proliferated in response to anti-CD40 monoclonal antibody. These findings imply that aberrantly activated B cells in PP undergo apoptosis independently of the CD95/CD95 L system during the course of infection with MAIDS virus.

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.

Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.

Evidence that the murine AIDS defective virus does not encode a superantigen

The T-cell receptor repertoire was analyzed in C57BL/6 mice upon infection with helper-free stocks of the pathogenic murine AIDS (MAIDS) defective virus in order to demonstrate if, as previously reported, this virus encodes a superantigen. A polyclonal T-cell stimulation involving T cells expressing multiple V beta subsets occurred within the first week of infection, while late in the disease we could note only a 50% deletion of V beta 5 CD8+ cells. Transfection of the MAIDS virus genomic DNA into fibroblasts and B cells expressing major histocompatibility complex class II molecules failed to show any stimulation of cells expressing the specific V beta (V beta 5) previously reported to respond to MAIDS virus-infected cells. In addition, mice lacking V beta 5 cells did not show any significant decrease in susceptibility to the disease compared with mice expressing V beta 5 and bred on the same genetic background. Our in vivo and in vitro results fail to demonstrate a role for a superantigen encoded by the MAIDS defective viral genome in the pathogenesis of MAIDS.

The role of TNFα on murine aids virus infection in glial cells

The role of TNFα on murine aids virus infection in glial cells

Lethal shock is inducible by lipopolysaccharide but not by superantigen in mice with retrovirus-induced immunodeficiency syndrome.

The retrovirus-induced murine AIDS (MAIDS) shares many features with human AIDS. Here, we examined the susceptibility of mice with MAIDS to staphylococcal enterotoxin-triggered shock. Following sensitization with D-galactosamine (D-Gal), mice with MAIDS were resistant to the otherwise lethal effect of superantigen staphylococcal enterotoxin A (SEA). Peak IL-2 levels in these mice after D-Gal/SEA challenge were 10-fold higher than those in uninfected controls, and concurrently, IL-10 levels rose markedly with reduction of circulating IL-1 and IFN-gamma. Treatment with neutralizing anti-IL-10 mAb before D-Gal/SEA challenge led to increased IFN-gamma levels in mice with MAIDS, and resulted in a dose-dependent mortality. In contrast, mice with MAIDS were more susceptible to the toxicity of bacterial endotoxin LPS than were uninfected controls. Administration of 100 micrograms LPS alone induced 50% lethality in mice infected with MAIDS virus 8 wk previously but not in uninfected controls. Administration of 10 micrograms LPS caused acute shock in D-Gal-sensitized mice with MAIDS. Peak TNF-alpha levels in these mice after LPS challenge were increased more than 10-fold, whereas IL-10 levels were one-third of those after SEA challenge. Moreover, serum IFN-gamma was undetectable in uninfected controls and rose to 1063 +/- 483 pg/ml in mice with MAIDS 4 h after LPS challenge. These results suggest that aberrant profiles of cytokine production are crucial in determining fatal outcome in these two types of septic shock in MAIDS.

The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus.

The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.

Molecular cloning and characterization of a murine AIDS virus-related endogenous transcript expressed in C57BL/6 mice.

The murine AIDS (MAIDS) virus has a unique sequence in the gag p12 region, which could be responsible for MAIDS development. RNA preparations from the spleens of normal uninfected C57BL/6 mice contain a transcript hybridizing with this sequence. Levels of the transcript in the kidney of C57BL/6 mice were higher than in the spleen, liver or thymus. Although BALB/c, NFS, DBA/2 and SL murine strains also contained genomic sequences hybridizing with the MAIDS virus-specific probe, no transcript hybridizing with the probe was detected in these strains of mice. The cDNAs carrying the transcript expressed in C57BL/6 mice were molecularly cloned. The complete nucleotide sequence of the clone indicates that the transcript is one of the endogenous murine leukaemia virus-related sequences containing large deletions from the R and U5 regions of the 5' long terminal repeat (LTR) to gag p15, from the C-terminal region of pol p40 (integrase) to the N-terminal region of env p15E, and many short deletions in the 3' LTR U3 region. The nucleotide sequence in the gag p12 region of the transcript was closely similar to that of the MAIDS virus, but the amino acid sequence was less similar because of frameshifting, even when translated. As the MAIDS virus was isolated from C57BL/6 mice with radiation-induced leukaemia, this transcript may be the progenitor of the MAIDS virus. To determine whether the gag p12 region of the transcript contains a functional sequence, a recombinant virus was generated by replacing the gag p12 region of a replication-competent BM5eco virus with that of the endogenous transcript. The recombinant virus was replication-competent, and the p12 region of the transcript retained the functional sequence present in the BM5eco virus.

Inhibition of murine AIDS (MAIDS), development by the transplantation of bone marrow cells carrying the Fv-4 resistance gene to MAIDS virus-infected mice

To examine whether the resistance allele of the Fv-4 gene (the Fv-4r gene) is a dominant inhibitory-product-encoding gene which an be used to prevent the development of murine AIDS (MAIDS), bone marrow cells from BALB/c-Fv-4wr mice were transplanted into BALB/c mice and C57BL/6 mice infected with MAIDS virus. Almost all of the virus-infected BALB/c and C57BL/6 mice developed MAIDS within 4 months and died 2 or 3 months later. However, when the virus-infected mice were subjected to cobalt irradiation and then given an intravenous injection of 10(7) BALB/c-Fv-4wr mouse bone marrow cells, the recipient mice survived much longer than the untreated mice, which suggests that the Fv-4 gene is a dominant inhibitory gene that is potentially useful in gene therapy of MAIDS.

Murine AIDS is initiated in the lymph nodes draining the site of inoculation, and the infected B cells influence T cells located at distance, in noninfected organs

The infection of cells which belong to the B-cell lineage is thought to be the primary event leading to the phenotypic and functional alterations seen in the murine AIDS (M. Huang, C. Simard, D. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). Using in situ hybridization, we studied the time course of the anatomic distribution of the murine AIDS-infected B cells in C57BL/6 mice inoculated intraperitoneally or in the foot pad with helper-free stocks of the defective murine AIDS virus. The local lymph nodes draining the injection site (the mediastinal or popliteal lymph nodes) were the primary organs in which infected B cells could be detected. From this initial site, the proliferating infected B cells were found to migrate progressively to most of the other lymph nodes and to the spleen. The bone marrow cells (containing the precursor B cells) were not found to be infected by the virus. These results suggest that the defective murine AIDS virus infects mature Ly-1- B cells present in lymph nodes. We compared the concanavalin A response of the T cells at an early time postinoculation, before all lymphoid organs are infiltrated with infected B cells. In lymphoid organs free of infected B cells, T cells were found to be hyperresponsive. In lymphoid organs in which infected B cells were present, T cells were hyporesponsive. These data suggest that infected B cells influence distant T cells, maybe by the release of a circulating factor or through another uninfected cell population activated by the infected B cells.