Koh Fujinaga

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells

Long‐term, persistent infection by HIV‐1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV‐1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV‐1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi‐quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV‐1‐seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus‐infected PBMC. These data are consistent with a mechanism of HIV‐1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3′-azido-3′-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.

Development of peptide vaccines inducing production of neutralizing antibodies against HIV-1 viruses in HLA-DQ6 mice

Peptide vaccines against HIV-1 were prepared according to the cassette theory that we had proposed previously. An amino acid sequence of B subtype consensus of the HIV-1 V3 region was introduced into the MHC binding component with a supermotif for various MHC class II. The peptide vaccines induced T-cell responses in the DQ6 mice in which only DQ6 molecules were expressed as MHC class II. By contrast, an original V3 peptide including the consensus sequence was non-immunogenic in the DQ6 mice. Antibodies obtained from the DQ6 mice immunized with the peptide vaccines neutralized laboratory B subtype strains of HIV-1 in vitro. It may be anticipated that these peptide vaccines protect infection of HIV-1 in DQ6 positive individuals.

SERIAL PASSAGE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1, GENERATES MISALIGNMENT DELETIONS IN NON-ESSENTIAL ACCESSORY GENES

Human immunodeficiency virus type 1 (HIV-1) derived from an infectious molecular clone pNL432 was extensively passaged in tissue culture by repeated rounds of acute infection. We previously showed the natural occurrence of a nonsense mutation in the vpr gene during continued passage of this virus. In this report, we show that two forms of large deletions (561 and 518 base pairs containing short direct repeats at the deletion junctions) occur after passage 50 in the region that spans the vif and vpr open reading frames. One model to explain the occurrence of these deletion regions is that such mutations result from misalignment of the growing point at a limited number of nucleotide positions. Infection of CD4+ T-cells with a recombinant HIV-1 construct containing the same vif to vpr deletion showed virtually no cytopathogenic phenotype. Thus, misalignment deletions at non-essential accessory genes of HIV-1 might be induced during replication, which result in the generation of virus with a low cytopathogenic potential.

A clearer distinction between HIV-1 paired isolates from peripheral blood mononuclear cells of asymptomatic carriers with and without CD8+ T-cells at nef rather than env V3 loci.

In asymptomatic carriers, the vast majority of human immunodeficiency virus type 1 (HIV-1) infection is non-productive whilst the clinical stage of disease is associated with significant virus expression. Virus-specific CD8+ T-cell functions are believed to play a major role in the generation of heterogeneous virus populations and in subsequent disease progression. Here, we prepared two types of HIV-1 isolate by culturing whole and CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from five asymptomatic carriers. The former is expected to be escape variant populations, whereas the latter would be mixed populations including the former viruses. The analyses of Nef and Env V3 sequence variations of viruses in a total of 77 and 44 DNA clones, respectively, allowed a direct comparison to be made of the differences between the paired isolates. Comparison of Nef sequences between the paired isolates showed them to be more distinct in two carriers with a relatively stable CD4/CD8 ratio (Nos 68 and 69), than in two other carriers with similar CD4/CD8 ratios (Nos 53 and 57), or in carrier No. 67, which had an extremely lower CD4/CD8 ratio. By contrast, a distinction between the paired isolates by use of the Env V3 sequences was only apparent in the latter three carriers. These results indicate that the predominant populations of HIV-1 in Nos 68 and 69 were sensitive to selective pressure from Nef-specific CD8+ T-cells, while those in Nos 53, 57, and 67 were sensitive to pressure from V3-specific CD8+ T-cells. It is noteworthy that Nos 53 and 57 progressed to an AIDS-related complex shortly and several years after this examination. These data suggest that HIV-1-induced pathogenesis is more strongly associated with the generation of variant nef alleles than with env V3 variants, and that these arise by CD8+ T-cell pressure.