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Mikako Fujita

Publications and source records attributed to Mikako Fujita.

At least 19 recordsLinked to original sources
PubMed2006

Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells.

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is caused in part by innate cellular factors such as apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) and TRIM5alpha, which restrict virus replication in monkey cells. Variant HIV-1 molecular clones containing both a 21-nucleotide simian immunodeficiency virus (SIV) Gag CA element, corresponding to the HIV-1 cyclophilin A-binding site, and the entire SIV vif gene were constructed. Long-term passage in a cynomolgus monkey lymphoid cell line resulted in the acquisition of two nonsynonymous changes in env, which conferred improved replication properties. A proviral molecular clone, derived from infected cells and designated NL-DT5R, was used to generate virus stocks capable of establishing spreading infections in the cynomolgus monkey T cell line and CD8-depleted peripheral blood mononuclear cells from five of five pig-tailed macaques and one of three rhesus monkeys. NL-DT5R, which genetically is >93% HIV-1, provides the opportunity, not possible with currently available SIV/HIV chimeric viruses, to analyze the function of multiple HIV-1 genes in a broad range of nonhuman primate species.

APOBEC-1 Deaminase↗
PubMed2006

Construction of gag-chimeric viruses between HIV-1 and SIVmac that are capable of productive multi-cycle infection.

Forty-nine recombinant viral clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus from the rhesus monkey (SIVmac), which carry chimeric gag (capsid/p2 region) genes in the background of the HIV-1 genome, were constructed to establish an HIV-1/monkey infection model system for human AIDS. Upon transfection, all the recombinants generated progeny virions at a level comparable to the parental HIV-1 clone and no major abnormalities were found in the virions, as examined by Western blot analysis. In infection experiments, 18 recombinants grew in human lymphocytic cells and six of these clones propagated as well as the parental virus, as monitored by virion associated-reverse transcriptase production. By contrast, none of the recombinants grew at a detectable level in monkey lymphocytic cells. The defective replication site(s) in human cells for non-infectious recombinants was mapped to the step before and/or during reverse transcription. Our results described here showed that HIV-1 type chimeric viruses between HIV-1 and SIVmac, which are capable of spreading productive infection, are readily constructed throughout the capsid/p2 region. In addition, it is suggested that there may be a viral determinant(s), other than Gag, responsible for the species-specific tropism of HIV-1 and which is associated with viral DNA synthesis.

Animals↗
PubMed2006

Morphological study on biologically distinct vpx/vpr mutants of HIV-2.

We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the vpx and vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.

Cell Line↗
PubMed2006

Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity.

We previously demonstrated that the expression in cells of human immunodeficiency virus type 1 (HIV-1) Vif is maintained at low level by proteasome-degradation. We examined the contribution of 16 lysines present in Vif (NL432 clone), which is composed of 192 amino acids (aa), to its expression within cells and to viral infectivity for non-permissive cells. To this end, various lysine-arginine mutations were introduced into wild-type (wt) Vif, and the mutational effects were monitored by transfection experiments. When all the lysines were changed to arginines, the mutant Vif was expressed in cells at much higher level than wt and was much more stable. Both N-terminal (aa nos. 34 and 36) and C-terminal (aa nos. 179 and 181) lysines were found to be almost sufficient for wt property. Different from this observation, one of the lysines at aa nos. 22 and 26 was demonstrated to be essential for the virus to grow in non-permissive cells. Our results showed that there is no clear co-relationship between the expression level of HIV-1 Vif and viral infectivity.

Amino Acid Sequence↗
PubMed2005

Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins.

The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.

Amino Acid Sequence↗
PubMed2005

Establishment of a biological assay system for human retroviral protease activity.

In order to obtain indicator cell lines that are exquisitely susceptible to human T-lymphotropic virus type 1 (HTLV-1), luciferase gene driven by HTLV-1 long terminal repeat (LTR) was transfected into lymphocytic H9 cells with neo gene, and cell lines were selected by G418. A cell line (H9/K30luc) was found to produce an extremely high level of luciferase only when co-cultured with HTLV-1 producer MT-2 cells. Both in the absence and presence of a reverse transcriptase (RT) inhibitor azidothymidine, H9/K30luc cells generated similarly high luciferase activity upon co-cultivation with MT-2 cells. To develop an equivalent system for human immunodeficiency virus type 1 (HIV-1), H9/NL432 cells, which are stably infected with HIV-1 and producing a low level of the virus-like MT-2 cells for HTLV-1, were generated. Together with the indicator cell line H9/H1luc for HIV-1 already reported, antiviral effects of some agents on HTLV-1 and HIV-1 could be readily and sensitively evaluated by similar methods. In fact, by using our system, an HIV-1 protease inhibitor, saquinavir, was demonstrated to be highly effective against HIV-1 but not against HTLV-1.

Antiviral Agents↗
PubMed2005

Unique characteristics of HIV-1 Vif expression.

We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.

Amino Acid Sequence↗
PubMed2004

High level expression of human immunodeficiency virus type-1 Vif inhibits viral infectivity by modulating proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site.

The human immunodeficiency virus type-1 Vif protein has a crucial role in regulating viral infectivity. However, we found that newly synthesized Vif is rapidly degraded by cellular proteases. We tested the dose dependence of Vif in non-permissive H9 cells and found that Vif, when expressed at low levels, increased virus infectivity in a dose-dependent manner. Surprisingly, however, the range of Vif required for optimal virus infectivity was narrow, and further increases in Vif severely reduced viral infectivity. Inhibition of viral infectivity at higher levels of Vif was cell type-independent and was associated with an accumulation of Gag-processing intermediates. Vif did not act as a general protease inhibitor but selectively inhibited Gag processing at the capsid and nucleocapsid (NC) boundary. Identification of Vif variants that were efficiently packaged but were unable to modulate Gag processing suggests that Vif packaging was necessary but insufficient for the production of 33- and 34-kDa processing intermediates. Interestingly, these processing intermediates, like Vif, associated with viral nucleoprotein complexes more rigidly than mature capsid and NC. We conclude that virus-associated Vif inhibits processing of a subset of Gag precursor molecules at the p2/NC primary cleavage site. Modulation of processing of a small subset of Gag molecules by physiological levels of Vif may be important for virus maturation. However, the accumulation of such processing intermediates at high levels of Vif is inhibitory. Thus, rapid intracellular degradation of Vif may have evolved as a mechanism to prevent such inhibitory effects of Vif.

CD4-Positive T-Lymphocytes↗
PubMed2004

Functional analysis of HIV-1 vif genes derived from Japanese long-term nonprogressors and progressors for AIDS.

We analyzed the function of human immunodeficiency virus type 1 (HIV-1) vif gene from Japanese long-term nonprogressors (LTNPRs) and progressors (PRs) for acquired immunodeficiency syndrome (AIDS). We constructed a basic HIV-1 infectious clone, which facilitated the incorporation and evaluation of vif from infected individuals. Proviral reporter clones carrying vif from six Japanese LTNPRs and seven PRs were then generated and their in vitro growth kinetics were analyzed. The vif clones, which could confer infectivity on reporter viruses, were considered active, and the ratio of the active clones to the number of clones examined per individual was determined. For the majority of LTNPRs, there was no correlation between presence or absence of functional vif with long-term nonprogression for AIDS. There was one exception in which all the clones examined had inactive vif, suggesting a probable association of inactive vif with the nonprogression. All PRs with high viral load had a high ratio of active vif clones. Our results suggest that the presence of functional vif would influence HIV-1 infectivity and disease progression in infected individuals.

Amino Acid Sequence↗
PubMed2004

Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation.

While the Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in non-permissive cells, it is rapidly degraded intracellularly. We have previously suggested that the rapid turn-over of Vif is biologically meaningful to prevent detrimental effects of this protein at high expression levels. We now studied the mechanism of Vif degradation by examining the blocking effect of protease inhibitors in pulse/chase experiments and by monitoring the extent of Vif ubiquitination. The rapid turn-over of Vif could be blocked by proteasome inhibitors, and Vif was highly ubiquitinated. Cytoskeletal Vif was found to be more stable than soluble cytosolic Vif. These degradation characteristics of Vif were cell type-independent and observed in both non-permissive and permissive cells. Characterization of a series of vif deletion mutants showed that amino acids predicted to be important for formation of beta-strand structures (amino acid nos. 63-70 and 86-89) were critical for maintaining a normal expression level of Vif and for viral infectivity. Finally, we performed comparative stability analysis of the four HIV-1 accessory proteins. Vif was unique in its short half-life and in the magnitude of the degradation. Taken together, we conclude that the proteasome degradation of HIV-1 Vif is a virologically important process and crucial for the function of Vif.

Amino Acid Sequence↗
PubMed2004

Role of Us3 gene of herpes simplex virus type 1 for resistance to interferon.

The sensitivity of a Us3-deletion mutant virus of herpes simplex virus type 1 (HSV-1) to consensus interferon (IFN) was compared with that of parental wild-type (wt) and the repaired viruses. Although one-step growth of the Us3-deficient virus in the IFN-treated HEp-2 cells was not markedly affected at a high multiplicity of infection (MOI), both the progeny virus yield and cytopathic effect were suppressed in a significantly higher degree in the mutant virus-infected cells than those in wt or the repaired virus-infected cells. This enhanced IFN-sensitivity of the mutant virus was more clearly demonstrated by the infection at a low MOI. In addition, both the size and number of plaques of the Us3-deficient virus in Vero cells were remarkably reduced with increasing concentrations of IFN, compared to those of wt or the repaired virus. These results indicate that the deletion of Us3 gene makes HSV-1 more sensitive to IFN.

Animals↗
PubMed2004

Determination of HIV-1 infectivity by lymphocytic cell lines with integrated luciferase gene.

We have established lymphocytic cell lines H9 and M8166 that contain integrated copy of luciferase gene under the control of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). While H9 is known to be non-permissive for or insensitive to some particular mutant strains of HIV/simian immunodeficiency virus (SIV), M8166 is one of the most susceptible lines to various HIV/SIVs. The luciferase gene driven by HIV-1 LTR was transfected into H9 and M8166 cells with the neo gene, and cell lines were selected by G418. The indicator cell lines thus obtained were designated H9/H1luc and M8166/H1luc, and monitored for their susceptibility to various HIV clones including in vitro-constructed mutants. Both cell lines, particularly M8166/H1luc, were found to be exquisitely sensitive to HIV-1 and HIV-2. Furthermore, they exhibited the response to infections by various viral clones exactly as expected from the characteristics of the original cell lines. These results indicated that our new indicator cell lines H9/H1luc and M8166/H1luc are eminently useful for a variety of molecular virological studies on HIV/SIV.

Cell Line↗
PubMed2004

Generation and characterization of HIV-1 clones chimeric for subtypes B and C nef.

The impact of human immunodeficiency virus type 1 (HIV-1) Nef on viral infectivity was evaluated by characterization of chimeric clones. Chimera with respect to the nef gene were constructed between subtypes B and C, and monitored for their replication in human peripheral blood mononuclear cells. The parental clones used were pNL432 (subtype B) and pIndie-C1 (Indian subtype C), which show considerable sequence heterogeneity in nef and distinct viral growth phenotype. While an enhancing effect of Nef on viral infectivity was noted, no significant growth difference was observed between the parental and chimeric clones. The difference in growth potential of the two subtype clones was mainly ascribable to viral sequence(s) other than nef. Our results here clearly showed that HIV-1 Nef does not significantly affect the in vitro viral infectivity in natural target cells.

Amino Acid Sequence↗
PubMed2003

Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells.

Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase.

Animals↗
PubMed2003

Amino acid residues 88 and 89 in the central hydrophilic region of human immunodeficiency virus type 1 Vif are critical for viral infectivity by enhancing the steady-state expression of Vif.

A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E(88)WRKKR(93)) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E(88) or W(89) residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E(88) and W(89) with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E(88) or W(89); a mutant with substitution of E(88) and W(89) with A; and a mutant with substitution of E(88), W(89), and R(90) with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E(88) and W(89) residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.

Amino Acid Sequence↗
PubMed2003

Susceptibility of HVS-immortalized lymphocytic HSC-F cells to various strains and mutants of HIV/SIV.

Susceptibility of HSC-F, a cynomolgus macaque cell line immortalized by Herpesvirus saimiri, to infection with various primate immunodeficiency viruses were monitored. While NL432 clone of human immunodeficiency virus type 1 (HIV-1) did not grow at all in HSC-F cells, GH123 and GL-AN clones of HIV-2, and MA239 clone of simian immunodeficiency virus isolated from macaque monkeys (SIVMAC) did grow in these cells. In addition, NM-3 clone of a chimeric simian and human immunodeficiency virus (SHIV) grew fairly well in HSC-F cells. Mutational analyses of accessory genes of GL-AN were successfully performed in the HSC-F cells. These results have thus demonstrated the importance of this cell line for molecular biological studies on HIV/SIV.

Animals↗
PubMed2003

[HIV-1, 2].

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Acquired Immunodeficiency Syndrome↗