Highly active antiretroviral therapy and beta-chemokines.
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Anti-HIV Agents↗
Biomedical subjects
B Brichacek
Publications and source records attributed to B Brichacek.
HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages.
Infection of macrophage lineage cells is a feature of primate lentivirus replication, and several properties of primate lentiviruses seem to have evolved to promote the infection of macrophages. Here we demonstrate that the accessory gene product Nef induces the production of two CC-chemokines, macrophage inflammatory proteins 1alpha and 1beta, by HIV-1-infected macrophages. Adenovirus-mediated expression of Nef in primary macrophages was sufficient for chemokine induction. Supernatants from Nef-expressing macrophages induced both the chemotaxis and activation of resting T lymphocytes, permitting productive HIV-1 infection. These results indicate a role for Nef in lymphocyte recruitment and activation at sites of virus replication.
Adenoviridae↗
Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.
After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex.
Cell Line↗
Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.
Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway.
CD4 Antigens↗
Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification.
The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.
Animals↗
A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.
X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.
Chromosomes, Artificial, Yeast↗
Quantitative competitive RNA PCR for quantitation of virion-associated HIV-1 RNA.
Quantitative competitive PCR is a highly sensitive technique that allows accurate quantitation of small amounts of RNA. We have modified the original method to include the use of an internal standard at all stages of sample analysis. In this way, the method can accommodate for variations in the recovery of viral particles and in the isolation of genomic RNA as well as provide a suitable competitive substrate during quantitative RNA PCR. We have used this method to characterize changes in virus load in plasma of HIV-1-seropositive individuals following their vaccination against opportunistic infections.
Blotting, Southern↗
Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM).
The vpr genes of human and simian immunodeficiency viruses (HIV/SIV) encode proteins which are packaged in the virus particle. HIV-1 Vpr has been shown to mediate the nuclear import of viral reverse transcription complexes in non-dividing target cells (e.g. terminally differentiated macrophages), and to alter the cell cycle and proliferation status of the infected host cell. Members of the HIV-2/SIV(SM) group encode, in addition to Vpr, a related protein called Vpx. Because these two proteins share considerable sequence similarity, it has been assumed that they also exhibit similar functions. Here, we report that the functions of Vpr and Vpx are distinct and non-redundant, although both proteins are components of the HIV-2/SIV(SM) virion and reverse transcription complex. Characterizing SIV(SM) proviruses defective in one or both genes, we found that Vpx is both necessary and sufficient for the nuclear import of the viral reverse transcription complex. In contrast, Vpr, but not Vpx, inhibited the progression of infected host cells from the G2 to the M phase of the cell cycle. Thus, two independent functions of the HIV-1 Vpr protein are encoded by separate genes in HIV-2/SIV(SM). This segregation is consistent with the conservation of these genes in HIV-2/SIV(SM) evolution, and underscores the importance of both nuclear transport and cell cycle arrest functions in primate lentivirus biology.
Animals↗
Regulation and characterization of the interferon-alpha present in patients with advanced human immunodeficiency virus type 1 disease.
To examine a possible association between plasma viremia and interferon-alpha (IFN-alpha) in patients with the acquired immunodeficiency syndrome (AIDS), we performed IFN plasma immunoadsorption by apheresis (IFN-alpha apheresis) in four volunteers with AIDS who had sustained levels of endogenous plasma IFN-alpha. IFN-alpha apheresis with two plasma volume exchanges was performed daily for 5 days. Clinical signs and symptoms and hematologic, virologic, and immunologic parameters were monitored. Two subjects developed anemia from phlebotomy, and one had a catheter++-associated bacteremia. The IFN-alpha apheresis was effective only in transiently removing IFN-alpha: depletion of IFN-alpha led only to its rapid reconstitution. Cell-associated HIV-1 was unchanged, but three of four subjects had a modest decrease in culturable plasma virus burden following the procedures. The recovery of in vivo HIV-1-related IFN-alpha by apheresis allowed its biologic and biochemical characterization. The HIV-1 IFN-alpha showed characteristics on ELISA, western blot, and biologic assays similar to two subspecies of the natural protein. The natural, recombinant, and HIV-1-induced IFN-alpha s demonstrated nearly identical antiviral activities. The HIV-1 IFN-alpha eluted from the column was not acid labile. The inability of large amounts of plasma IFN-alpha found in some patients with AIDS to affect viral burden likely reflects properties of the virus or of host factors independent of IFN-alpha.
Acquired Immunodeficiency Syndrome↗
Increased plasma human immunodeficiency virus type 1 burden following antigenic challenge with pneumococcal vaccine.
Primary factors that influence virus burden during human immunodeficiency virus type 1 (HIV-1) disease progression remain a fundamental issue in pathogenesis. Because pneumococcal vaccine is routinely given to HIV-1-infected patients and replication of HIV-1 within CD4 T cells is dependent on the activation state of the cell, it was investigated whether the T cell activation that enhances the immune response to vaccines may also enhance HIV-1 replication. Vaccination of asymptomatic HIV-1-infected patients led to rapid and significant increases in virus burden in some patients. The magnitude of these increases correlated significantly with the extent of the antibody response to the vaccination. Thus, antigenic stimulation by vaccines designed to prevent secondary infections may promote HIV-1 replication in certain patients. These findings provide a window for examining HIV-1 pathogenesis and for determining the appropriate preventive measures against other diseases in HIV-1-infected persons.
Adult↗
Molecular basis of cell cycle dependent HIV-1 replication. Implications for control of virus burden.
Research is beginning to yield insight into determinants which govern cell cycle dependence of provirus establishment by the onco-retroviruses. In the case of HIV-1, nucleophilic components associated with the viral preintegration complex facilitate mitosis independent nuclear localization of viral DNA and provirus establishment. Differences in the metabolic activity between G0 T cells and macrophages, the two primary targets for HIV-1 infection, lead to significantly different outcomes with regards to provirus establishment following infection of these cells. Thus, macrophages appear fully permissive to productive HIV-1 replication while non-dividing (G0 T cells) restrict virus replication at a step which proceeds nuclear import of viral DNA. The requirement for T cell activation in productive HIV-1 replication has important implications for the relationship between immune activation and virus burden. It remains to be determined whether modulating the immune activation status of the infected individual may provide an opportunity for modulating virus burden and influencing disease course.
Acquired Immunodeficiency Syndrome↗
X-linked lymphoproliferative disease.
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Bone Marrow Transplantation↗
Early activation of the proto-oncogene c-fgr during Epstein-Barr virus immortalization.
In an attempt to clarify the chronological relationships between Epstein-Barr virus (EBV) infection, B cell immortalization and c-fgr activation, we evaluated for the presence of EBV-determined nuclear antigen (EBNA), cellular DNA synthesis and expression of c-fgr-specific RNA following infection of human peripheral blood lymphocytes with B95-8 EBV. High expression of c-fgr was observed prior to EBNA detection and cellular DNA synthesis in EBV-infected cells. These results suggest that activation of c-fgr is an essential event during the early phase of EBV immortalization.
Antigens, Viral↗
HIV-1 infectivity of human carcinoma cell lines lacking CD4 receptors.
Human immunodeficiency virus type 1 (HIV-1) prototype, HIV1 LAV, and a Zairian virus HIV1 NDK, an isolate highly cytopathic for CD4+ lymphocytes, were used to infect eleven different CD4 negative non-lymphoid human cell lines. Eight of the lines were derived from carcinomas wherein human papillomavirus was thought to have been etiologic. All these cell lines lacked CD4 receptor and CD4 specific mRNA. After cocultivation with sensitive CEM cells, HIV-1 LAV was rescued from six infected cell lines and HIV-1 NDK from nine. Shedding of free virus into the culture medium was observed in three cell lines infected by HIV-1 NDK and in only one cell line infected by HIV-1 LAV. The infectibility of CD4 negative cell lines indicates that both HIV-1 strains were able to use a CD4 independent mechanism to infect the cells; however, HIV-1 NDK showed the higher efficiency of infection. This virus was also able to overcome the intracellular block of viral reproduction. These results suggest that a broader spectrum of cell types of non-lymphoid origin lacking the CD4 receptor can serve as a viral reservoir. In some cases they are direct producers of infectious HIV-1 particles. This suggests, that in addition to immunosuppressive mechanisms, HIV-1 could play a more direct role in induction of neoplastic changes.
CD4 Antigens↗
HIV1 cytopathogenicity-genetic difference between direct cytotoxic and fusogenic effect.
Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spel1042/EcoRl4183, containing the 3'-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIV1 NDK was associated with the simultaneous presence of HIV1 NDK fragments BssHll255/Spel1042 and EcoRl5278/Xhol8401 which contained the splicing donor, packaging sequence, p18 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIV1 NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments BssHll/Spel and EcoRl/Xhol were by themselves necessary but not sufficient to induce formation of large syncytia.
Cell Death↗
The human immunodeficiency virus (HIV) gag gene product p18 is responsible for enhanced fusogenicity and host range tropism of the highly cytopathic HIV-1-NDK strain.
Formation of large syncytia and rapid cell killing are characteristics of the Zairian human immunodeficiency virus type 1 isolate HIV-1-NDK, which is highly cytopathic for CD4+ lymphocytes in comparison with the HIV-1-LAV prototype. Chimeric viruses containing different combinations of HIV-1-NDK genetic determinants corresponding to the splice donor, the packaging signal, and the coding sequence of the p18gag protein together with the HIV-1-NDK EcoRI5278-XhoI8401 fragment were obtained by polymerase chain reaction-directed recombination. Phenotypic analysis of recombinant viruses indicated that 75 amino acids from the N-terminal part of HIV-1-NDK p18gag protein together with the HIV-1-NDK envelope glycoprotein are responsible for enhanced fusogenicity of HIV-1-NDK in CD4+ lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4- cells lines. The HIV-1-NDK splice donor/packaging sequence and the sequence encoding the gag protein p25 were not important for the variation observed in HIV-1 fusogenicity.
Amino Acid Sequence↗
Immunohistologic identification of Epstein-Barr virus-induced hepatitis reactivation after OKT-3 therapy following orthotopic liver transplant.
Reactivated Epstein-Barr virus infection associated with hepatitis appeared in a liver transplant patient receiving monoclonal OKT-3 antibody for rejection. The histologic findings in liver biopsy specimens characteristic of allograft rejection were observed prior to and during the initial phase of antirejection therapy. However, failure of a complete response to antirejection therapy promoted rebiopsy. The specimen showed portal infiltrates composed predominantly of plasma cells and immunoblasts. The presumptive diagnosis of Epstein-Barr virus hepatitis was confirmed by staining frozen liver tissue for Epstein-Barr virus nuclear-associated antigen. OKT-3 therapy was discontinued, and cyclosporine and steroid doses were reduced. Gradually, clinical features, serum aminotransferase and bilirubin levels, and the portal lymphoid infiltrate resolved. Epstein-Barr virus serology showed an increase in convalescent titers IgG-antiviral capsid antigen, and Epstein-Barr virus nuclear-associated antigen. The histologic, clinical, and laboratory features supporting the diagnosis of Epstein-Barr virus hepatitis in a liver transplant patient are presented and discussed. This diagnosis guided appropriate therapy.
Antibodies, Monoclonal↗
Hepatitis in fatal infectious mononucleosis.
A detailed clinicopathologic analysis of 30 patients with sporadic fatal infectious mononucleosis and 31 males with fatal infectious mononucleosis and the X-linked lymphoproliferative syndrome was performed to determine the extent of hepatic dysfunction in these cases. At death, the median age of patients with sporadic infectious mononucleosis was 10.7 yr vs. 2.4 yr for X-linked lymphoproliferative syndrome. The median survival time was 8 wk for sporadic infectious mononucleosis and only 4 wk for X-linked lymphoproliferative syndrome. The male to female ratio was 3:2 in sporadic infectious mononucleosis; all patients with X-linked lymphoproliferative syndrome were males. Fever, sore throat, lymphadenopathy, hepatomegaly, and splenomegaly were prominent findings. Hepatic dysfunction was uniformly present and caused death in 13 of 30 sporadic infectious mononucleosis cases and 18 of 31 X-linked lymphoproliferative syndrome cases. Diagnosis of infectious mononucleosis was confirmed by heterophile antibody titers or Monospot, Epstein-Barr virus antibody studies, viral culture, molecular hybridization studies, clinical and histologic findings, and pedigree analysis.
Child↗