Extensive variation of human immunodeficiency virus type-1 in vivo

Genotypic variation among independent isolates of human immunodeficiency virus type-1 (HIV-1) is well known, but its molecular basis and biological consequences are poorly understood. We examined the genesis of molecular variation in HIV-1 by sequential virus isolations from two chronically infected individuals and analysis of recombinant HIV-1 genomic clones. In three different virus isolates full-length HIV-1 clones were identified and found to consist, respectively, of 17, 9 and 13 distinguishable, but highly-related, viral genotypes. Thirty-five viral clones derived from two HIV-1 isolates obtained from the same individual but 16 months apart showed progressive change, yet were clearly related. Similar changes in the HIV-1 genome did not occur in vitro during virus isolation and amplification. The results indicate that HIV-1 variation in vivo is rapid, that a remarkably large number of related but distinguishable genotypic variants evolve in parallel and coexist during chronic infection, and that 'isolates' of HIV-1, unless molecularly or biologically cloned, generally consist of complex mixtures of genotypically distinguishable viruses.     

天花粉蛋白抗人免疫缺陷病毒1型研究

目的检测用基因工程方法制备的天花粉蛋白抗人免疫缺陷病毒1型的活性。方法将人免疫缺陷病毒1型在细胞系中培养,加入天花粉蛋白后,观察细胞病变情况,检测P24抗原及逆转录酶的活性。结果加入天花粉蛋白的细胞始终未见细胞病变,P24抗原及逆转录酶检测均为阴性;阴性对照组细胞在第7 d以后均出现人免疫缺陷病毒1型感染典型细胞病变,P24抗原及逆转录酶检测均为阳性;阳性对照组始终未见HIV-1感染典型细胞病变,逆转录酶的活性检测和P24抗原检测结果均为阴性。结论天花粉蛋白在培养细胞系中可以有效地抑制人免疫缺陷病毒1型。     

Acquisition of the human adeno-associated virus type-2 rep gene by human herpesvirus type-6.

Human herpesvirus type-6 (HHV-6) is a recently isolated herpesvirus which is highly prevalent in adult populations around the world. HHV-6 was first isolated from the peripheral blood of six individuals with lymphoproliferative disorders, two of whom were also infected with human immunodeficiency virus. HHV-6, in common with other herpesviruses, transactivates the HIV long terminal repeat linked to reporter genes and has in addition been shown to accelerate HIV gene expression and CD4 cell death in cultures co-infected with both viruses. The virus is tropic for CD4+ lymphocytes and persists in the peripheral blood of most seropositive individuals. We have now identified a gene in HHV-6 encoding a 490-amino-acid polypeptide homologous to the human adeno-associated virus type-2 (AAV-2) rep gene. This gene has an essential role in AAV-2 DNA replication, can trans-regulate homologous and heterologous gene expression, and inhibits cellular transformation. The acquisition of rep by HHV-6 could be due to natural transfer of genetic information between DNA viruses of eukaryotes and is likely to have important consequences for the life-cycle of HHV-6 and for the host CD4 cell.     

Soluble CD4 molecules neutralize human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) infection can bring about total collapse of the immune system by infecting helper T lymphocytes which express CD4, the molecule which mediates interaction between the cell surface and viral envelope glycoprotein gp120 (refs 3-10). HIV apparently escapes the effects of neutralizing antibodies in vivo by generating new variants which must still interact with CD4 to maintain a cycle of infection. One route to block HIV infection, therefore, could use solubilized CD4 protein to inhibit attachment of the virus to its target cell. We have used recombinant DNA techniques to generate soluble forms of CD4, and show here that these are potent inhibitors of HIV infection in vitro.     

Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.     

Infection of rabbits with human immunodeficiency virus

An important requirement for the development of a vaccine against the human immunodeficiency virus (HIV-1), the causative agent of AIDS, is a readily available animal model that would allow possible immunogens to be evaluated. The only species to have been infected with HIV-1 so far is the chimpanzee. However, the scarcity of this animal and its designation as an endangered species place severe restrictions on its use as an animal model. Attempts to infect mice, rats, hamsters, guinea-pigs, musk shrews, and rabbits with HIV-1 or infected cells have all been unsuccessful. We now report that the intraperitoneal inoculation of rabbits with HIV-1 or chronically infected H9 cells consistently induces a persistent infection.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated virus

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cosl4 and cos56, which represents the HSV-1 genome was used for generation of this rHSV.Rep andcap genes of AAV-2 were inserted intoXba I site ofUL2 gene on cod, generating cos6rcΔUL2. After being digested withPac I, cos6-rcΔUL2 and the other 4 cosmids were cotransfected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carryingrep andcap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence ofrep andcap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Sequence of simian immunodeficiency virus and its relationship to the human immunodeficiency viruses

The characterization of HIV-1 (HTLV-III/LAV), the human retrovirus associated with AIDS (acquired immune deficiency syndrome) has led to the identification of a group of related human and simian retroviruses which also infect CD4-bearing T lymphocytes. Simian T-lymphotropic virus type III (simian immodeficiency virus) from macaques (STLV-IIIMAC) induces symptoms similar to those of AIDS in infected macaques, but isolates from African green monkeys (STLV-IIIAGM) and mangabeys (STLV-IIMM) appear to be non-pathogenic in these animals. A human virus immunologically related to STLV-IIIAGM (HTLV-IV), reported to have been isolated from healthy humans, has been shown to be almost identical to STLV-IIIAGM, which has called into question the independent origin of these viruses. Here we report the complete DNA sequence of STLV-IIIAGM and analyse its relationship with the genomes of the HTLV-IIIB strain of HIV-1, HIV-2ROD (previously called LAV-2) and several ungulate lentiretroviruses. STLV-IIIAGM and HIV-2 are closely related, and more distantly related to HIV-1.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.     

Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.