Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Single amino-acid changes in HIV envelope affect viral tropism and receptor binding

Infection by the human immunodeficiency virus (HIV) is initiated by the binding of its extracellular envelope glycoprotein, gp120, to the CD4 antigen on target cells. To map the residues of the HIV-1 glycoprotein that are critical for binding and to analyse the effects of binding on viral infectivity, we created 15 mutations in a region of gp120 that is important for binding to CD4 (refs 4,5). We find that substitution of a single amino acid (tryptophan at position 432) can abrogate CD4 binding and that virus carrying this mutation is non-infectious. By contrast, other amino-acid changes in the same region do not affect CD4 binding but restrict viral tropism: virions containing isoleucine substitutions at position 425 lose their ability to infect a monocyte cell line (U937 cells) but can still infect T-lymphocyte cell lines (CEM, SUP-T1) and activated human peripheral blood lymphocytes. These results indicate that cellular tropism of HIV can be influenced by a single amino-acid change in gp120.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor

The main receptor for the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) on T and B lymphocytes, monocytes and macrophages is the CD4 antigen 1-3. Infection of these cells is blocked by monoclonal antibodies to CD4(1,2) and by recombinant soluble CD4(4-9). Expression of transfected CD4 on the surface of HeLa and other human cells renders them susceptible to HIV infection 10. HIV-antibody complexes can also infect monocytes and macrophages by means of receptors for the Fc portion of immunoglobulins (FcR)11-13), or complement receptors 14,15. The expression of IgG FcRs can be induced in cells infected with human herpes viruses such as herpes simplex virus type 1 (HSV-1)16,17 and human cytomegalovirus (CMV)18-21. Here we demonstrate that FcRs induced by CMV allow immune complexes of HIV to infect fibroblasts otherwise not permissive to HIV infection. Infection was inhibited by prior incubation with human IgG, but not by anti-CD4 antibody or by recombinant soluble CD4. Once HIV had entered CMV-infected cells by means of the FcR, its replication could be enhanced by CMV transactivating factors. Synergism between HIV and herpes viruses could also operate in vivo, enhancing immunosuppression and permitting the spread of HIV to cells not expressing CD4.     

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4

Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection.     

HIV infection is blocked in vitro by recombinant soluble CD4

The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome. HIV recognition of CD4 is probably mediated through the virus envelope glycoprotein (gp120) as shown by co-immunoprecipitation of CD4 and gp120 (ref.5) and by experiments using recombinant gp120 as a binding probe. Here we demonstrate that recombinant soluble CD4(rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.     

The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kappaB activity. Knockdown of Murr1 increased NF-kappaB activity and decreased IkappaB-alpha concentrations by facilitating phospho-IkappaB-alpha degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.     

Accessory cell-dependent selection of specific T-cell functions

Activation of many T-cell functions depends on their interaction with antigen-presenting accessory cells which express I region associated (Ia) products. Cells expressing accessory cell function include those of the monocyte/macrophage lineage and dendritic cells. More recently, B cells and a number of tumour cell lines of macrophage or B-cell origin were shown to act as accessory cells in certain assays. We showed previously that normal peritoneal exudate macrophages (PEC) induced both T-cell proliferation as well as T-cell help, whereas various Ia+ tumour lines of macrophage or B-cell origin, although stimulating antigen-specific T-cell proliferation, did not significantly activate T-cell help. We report here that during the initial T-cell activation in vitro accessory cells (PEC or Ia+ tumour cells) select particular T cells to express previously determined functions. Moreover, some tumour cell lines induce suppressor T cells which inhibit helper activity.     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14,and the susceptibility of the cells to HIV-1 is then changed.Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion.Based on these studies,we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937.Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937,while another CX- CR4-specific mAb B-R24 did not show any effect on this binding.On the other hand,two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4),which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14- specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4.These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14, and the susceptibility of the cells to HIV-1 is then changed. Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion. Based on these studies, we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937. Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937, while another CXCR4-specific mAb B-R24 did not show any effect on this binding. On the other hand, two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4), which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14-specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4. These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

反式激活HIV-1 LTR的HHV-6基因片段B701的缺失研究

ＨＨＶ－６在体外可以激活ＨＩＶＬＴＲ引导的基因表达［１］，其基因组中一基因片段Ｂ７０１已被证明与这种激活作用有关［２］．Ｂ７０１编码１４３个氨基酸的蛋白质，可与ＨＨＶ－６基因组中其它基因片段协同作用提高对ＨＩＶＬＴＲ的激活效力．对Ｂ７０１进行缺失突变，得到突变体ＲＳＶ－Ｂ７０１－ｄ１（３′端缺失１８１个ｂｐ）、ＲＳＶ－Ｂ７０１－ｄ６（３′端缺失３５３个ｂｐ），将这两个缺失突变体分别与ＨＩＶ－Ｌｕｃ共转染受体细胞，通过ＬＵＣ活性分析发现，缺失突变体ｄ１、ｄ６不但仍具有激活能力，而且ｄ６的激活能力要远远大于ｄ１．二级结构预测初步揭示了Ｂ７０１对ＨＩＶ－ＬＴＲ的反式激活作用机制，为阐明ＨＨＶ－６基因产物与ＡＩＤＳ的病理关系提供了依据．