AIDS is a progressive disease associated with steady loss of helper T cells and several other functions. As the disease evolves, cytopathogenic human immunodeficiency (HIV) variants of increasing virulence can be isolated from the host. The HIV is an unusually variable genome by virtue of a low replication fidelity. In this report we describe our effort to test the hypothesis that there is a correlation between virus variability and cytopathogenicity, and further, that there is an "impact" of the virus infection on the expression of host cellular genes. To search for such a relationship, we infected H-9 cells (human CD4+ lymphoblastoid cell line) with each of 5 isolates of HIV of distinct origin and cytopathogenicity. To measure the influence of the virus infection on the expression of host cellular genes, shortly after infection, (3 h or 13 h), cells were radiolabeled and the radioactive polypeptides separated by two-dimensional gel electrophoresis. Radiofluorographs were prepared and analyzed to determine relative rates of biosynthesis of cellular polypeptides. To organize the large amounts of data found, cluster analysis and principal component analysis were used to expose the data in formats that allowed a model construction. The rates of biosynthesis of many cellular polypeptides were altered upon viral infection in terms of both enhancements and impairment of biosynthesis. Some of the variation in polypeptide synthesis was isolate-specific, while most alterations were of modest magnitude. There appears to be no "overall effect" associated with infection by a cytopathic variant of the virus. Polypeptides affected by the cytopathic variants were determined as targets for further investigation. The method used promotes the measurement of "ensemble" information that is characteristic of the process and it promotes the creation of models of virus action.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

Using two-dimensional gel electrophoresis to study immune response against intracellular bacterial infection.

Principal component analysis was applied to two-dimensional (2-D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so-called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2-D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.

Animals↗

PubMedDate not supplied

Proteomic analysis of T cell activation in the presence of cyclosporin A: immunosuppressor and activator removal induces de novo protein synthesis.

Cyclosporin A (CsA), a fungal metabolite used in organ transplantation, blocks the immune responses by interfering with early activation signals preventing the induction of the IL2 gene. We have previously reported that the removal of the immunosuppressor provokes the transcription of the IL2 encoding gene. We have now investigated whether the transcription and translation of other genes accompanies this process. Withdrawal of CsA and Concanavalin A (ConA) from cultures of murine T cells activated by ConA in the presence of CsA leads to substantial changes in the pattern of radio-labelled proteins. A large number of polypeptides were synthesised de novo. In addition, a set of polypeptides detected prior to immunosuppressor elimination was not anymore synthesised. Finally, besides these qualitative changes, quantitative differences in terms of increased or decreased polypeptide abundance were also observed. The results demonstrate that activation in the presence of CsA has programmed the T cells to transcribe and translate a large number of genes, without further reactivation, once the immunosuppressor and the activator were removed.

Animals↗

PubMedDate not supplied

An attempt to identify gene products related to the induction of an antiviral state in macrophages resistant and sensitive to IFN-gamma.

The activation of bone-marrow-derived macrophages by IFN-gamma (IFN gamma) partially inhibits mouse hepatitis virus 3 (MHV3) replication only in cells from resistant A/J mice, and not in cells originating from susceptible BALB/c mice. The computer image analysis of gels obtained from 2D-SDS-PAGE of extracted proteins of IFN gamma-activated A/J or BALB/c macrophages enabled us to identify and tag several gene products that were synthesized at elevated or diminished levels. Comparisons of the patterns of non-activated and IFN gamma-activated A/J macrophages revealed 3 gene products which increased, 1 which newly appeared, 6 which decreased and 20 which disappeared upon IFN gamma activation. The protein pattern of BALB/c macrophages revealed 13 gene products which increased, 8 which decreased and 8 which disappeared in IFN gamma-activated BALB/c macrophages. Whether these proteins are involved in the induction of an antiviral state against MHV3 growth remains to be investigated. Macrophages from mice with different genetic background (A/J and BALB/c), upon IFN gamma activation, behave differently at a molecular level, and this observation is consistent with their distinct expression of antiviral state against MHV3.

Animals↗

PubMedDate not supplied

The pattern of proteins synthesized in the liver is profoundly modified upon infection of susceptible mice with mouse hepatitis virus 3.

Susceptible BALB/c mice, after experimental infection with mouse hepatitis virus 3 (MHV3), revealed virus titres in the liver that increased gradually to a peak of 8 x 10(5) PFU/g of tissue after 3 days' infection, when the mice died of acute hepatitis. BALB/c mice were infected with MHV3, subsequently labelled in vivo with 35S-methionine, and then the liver preparations from both infected and non-infected animals were subjected to two-dimensional gel electrophoresis. Comparisons of the patterns by computer image analysis revealed 17 gene products which increased, and 8 gene products which decreased, upon virus infection in their two-dimensional gel spot intensity. We conclude that during MHV3 infection of a susceptible strain of mice, a major modification in protein synthesis occurs. The pattern alterations were not related to the virus gene products but were mostly endogenous mouse proteins. Whether these proteins are a result of a defence attempt by the animal, or are dictated by the virus in order to prevent a protective response from happening, remains to be shown.

Animals↗

PubMedDate not supplied

Molecular complexity of Qa-2 antigens demonstrated by two-dimensional gel electrophoresis.

Biosynthetically labelled Qa-2 antigens were isolated from mouse spleen cells by immunoprecipitation with anti-Qa-2 antisera. When newly synthesized Qa-2 molecules from several different inbred strains were analysed by two-dimensional (2D) gel electrophoresis; four different phenotypes were observed that differed in the number of polypeptides present. The ability to distinguish Qa-2+ phenotypes was used to map the recombination points in two congenic strains, B6.Tlaa and A.Tlab. No alternative Qa-2-like polypeptides were detected in B6.K1 (Qa-2-) cells using a polyspecific rabbit antiserum against mouse class I antigens, but a new molecule was detected in BALB/cBy (Qa-2-) cells. Pulse-chase and surface-labelling experiments showed that some, but not all, of the newly synthesized Qa-2 precursor forms are processed to mature cell surface molecules.

Animals↗