Meiko Kawamura

Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.

Function of human immunodeficiency virus type 1 Vpu protein in various cell types.

We evaluated the function of human immunodeficiency virus type 1 vpu gene in various cell lines. We established a highly sensitive system consisting of chloramphenicol acetyltransferase and reverse transcriptase assays and used it to monitor the effects of mutation of the vpu gene. In some cell lines, Vpu protein was not required at the early phase of viral replication but was important for efficient virion production. In these cells, the Vpu protein functioned effectively irrespective of the presence of intact env gene products. Likewise, CD4 gene expression had no effects on Vpu function. In the other cell lines tested, Vpu protein was not important for virion release, and the vpu mutant clone generated a normal level of progeny virions upon transfection.

Establishment of a phylogenetic survey system for AIDS-related lentiviruses and demonstration of a new HIV-2 subgroup.

We designed a universal primer (UNIPOL) for DNA amplification of AIDS-related viruses. The phylogenetic tree constructed from the presumed sequences amplified with UNIPOL was representative of the tree calculated from whole pol gene sequences so far reported. UNIPOL was able to amplify the sequences of all four major groups of primate lentiviruses and also that of a distinct virus from a Ghanaian patient with an AIDS-related complex, designated GH-2. This strain scarcely hybridizes with known HIV/simian immunodeficiency virus (SIV) DNA probes. Sequence analysis of the only amplified fragment revealed rapidly that GH-2 was quite similar to the recently reported HIV-2ALT(D205) and that these two viruses form a new subgroup distint from known HIV-2 and SIVmac/SIVsm in the large HIV-2 group. This system will be useful for further phylogenetic study of various primate lentiviruses.

Growth ability of human immunodeficiency virus type 1 auxiliary gene mutants in primary blood macrophage cultures.

A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif, vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.

Human Immunodeficiency Virus Vpx Is Required for the Early Phase of Replication in Peripheral Blood Mononuclear Cells

Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild‐type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx‐minus virus was detected in primary macrophage cells. A highly sensitive single‐round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication‐defect at a late phase in primary cells of the vpx mutant was obtained by a transfection‐coculture method. These results indicate that the virion‐associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.

Biological characterization of human immunodeficiency virus type 1 and type 2 mutants in human peripheral blood mononuclear cells

SummaryMutants of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), which have been shown to be infectious in established cell lines, were tested for ability to replicate and induce syncytium formation in human peripheral blood mononuclear cells (PBMC). Thevpu mutant of HIV-1 showed depressed kinetics of replication in an established T cell line, as reported previously, but in PBMC, its replication was similar to that of the wild type virus. Thevpx gene of HIV-2 was required for efficient virus propagation in PBMC, but not in an established T cell line, as previously reported. However, the growth rates of thevpx mutant in PBMC preparations from two individuals were different. The results of experiments on infection of PBMC with thevif andvpr mutants of HIV-1 and HIV-2 were essentially consistent with previous results of infection of established T cell lines. No negative effect of thenef gene products of HIV-1 and HIV-2 was observed. The abilities of the wild type virus and the mutants of HIV-1 to induce syncytium formation in both PBMC and established cell lines were similar. In contrast, neither the wild type nor any of the mutants of HIV-2 induced syncytium formation in PBMC. These results suggest that the functions of some genes can be detected only in mixed populations or primary cells such as PBMC. Studies on the roles of these genes in PBMC may provide a better understanding of their functions in vivo.

Isolation and characterization of simian immunodeficiency virus from African white-crowned mangabey monkeys ( Cercocebus torquatus lunulatus)

SummaryForty-eight of 236 sera from seven species of African non-human primates in Kenya, including those of white-crowned mangabey monkeys (Cercocebus torquatus lunulatus) had antibodies to simian immunodeficiency viruses (SIVs). Isolates of simian lentivirus were obtained from seropositive white-crowned mangabey monkeys which are indigenous in West Africa. This virus, designated as SIVWCM, appeared morphologically similar to HIV by electron microscopy, showed Mg2+-dependent reverse transcriptase activity, and induced cytopathic effects in human CD 4-positive cells. Western blotting analysis revealed thatenv products of SIVWCM cross-reacted with those of SIVAGM more strongly than with those of HIV-1 and SIVMAC, and clear hybridization bands were detected with an SIVAGM probe. For comparison of the virus sequence with those of other primate lentiviruses, part of thepol gene and the long terminal repeats (LTRs) were amplified and cloned. Sequencing showed that SIVWCM isolates were closely related to SIVAGM isolates. This study suggested that SIVAGM from theCercopithecus genus and SIVWCM from theCercocebus genus may be members of an SIV group that is genetically distinct from the SIV from a sooty mangabey monkey (SIVSMM) of the genusCercocebus, to which the white-crowned mangabey monkey also belongs.

HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases

In‐frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV‐1) gag gene, and potentials of the mutants to suppress the replication of wild‐type HIV‐1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV‐1 replication completely in single‐round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV‐1 reported to date, and to be effective at both early and late viral replication phases. T‐cells, which are engineered to express the C6b Gag in response to HIV‐1 infection, were perfectly resistant to HIV‐1.

Compatibility of rev gene activity in the four groups of primate lentiviruses.

The compatibility of rev genes derived from various primate immunodeficiency viruses of all distinct subgroups identified was assessed in three experimental systems: complementation experiments between proviral rev and gag mutants, evaluation of the ability of the rev gene products to activate proviral reporters, and examination of the capacity of various viruses to augment marker gene expression in the infected reporter cell lines. In all systems, human immunodeficiency virus type 1 (HIV-1) rev was not substantially substituted or was extremely poorly substituted by the rev of the other viruses. The rev of simian immunodeficiency virus (SIV) from a mandrill could be exchanged by HIV-1 rev. In contrast, the rev gene products of all viruses efficiently activate HIV-2 and SIV from an African green monkey.

Functional analysis of long terminal repeats derived from four strains of simian immunodeficiency virus SIVAGM in Relation to Other Primate Lentiviruses

The promoter activity of long terminal repeats (LTRs) of four strains of the simian immunodeficiency virus isolated from African green monkeys (SIVAGM) was compared with those of various LTRs derived from the other representative primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), SIV from a rhesus monkey (SIVMAC), and SIV from a mandrill (SIVMND). The expression of the LTRs was evaluated by monitoring chloramphenicol acetyltransferase production after transfection of reporter plasmid clones. In the absence of viral tat, all SIVAGM LTRs acted as much more efficient promoters than any of the other LTRs. When tat gene products were supplied in trans, LTRs of SIVAGM and SIVMND were activated inefficiently relative to high responder LTRs of HIV-2 and SIVMAC. The LTR of HIV-1 was highly activated by HIV-1 tat, but not so much by HIV-2, SIVAGM, and SIVMND tat.