Akiko Sakurai

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.

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

ABSTRACT 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 (E88WRKKR93) 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 E88 or W89 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 E88 and W89 with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E88 or W89; a mutant with substitution of E88 and W89 with A; and a mutant with substitution of E88, W89, and R90 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, E88 and W89 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.

Cyclophilin A-Independent Replication of a Human Immunodeficiency Virus Type 1 Isolate Carrying a Small Portion of the Simian Immunodeficiency Virus SIVMAC gag Capsid Region

ABSTRACT Hybrid viruses between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus strain mac (SIVMAC) are invaluable to various fields of HIV-1 research. To date, however, no replication-competent HIV-1 strain containing the gagcapsid (CA) region of SIVMAC has been reported. To obtain the viable gag gene chimeric virus in an HIV-1 background, seven HIV-1 strains carrying a part of SIVMAC CA or a small deletion in the CA region were constructed and examined for their biological and biochemical characteristics. While all the recombinants and mutants were found to express Gag and to produce progeny virions on transfection, only one chimeric virus, which has 18 bp of SIVgag CA sequence in place of the region encoding the HIV-1 CA cyclophilin A (CyPA)-binding loop, was infectious for human cell lines. Although this chimeric virus was unable to grow in monkey lymphocytic cells like wild-type (wt) HIV-1 did, it grew much better than wt virus in the presence of cyclosporin A in a human cell line which supports HIV-1 replication in a CyPA-dependent manner. These results indicate that the transfer of a small portion of the SIVMAC CA region to HIV-1 could confer the CyPA-independent replication potential of SIVMAC on the virus.

Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype.

Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.

Analysis of the cell-dependent replication potentials of human immunodeficiency virus type 1 vif mutants

Eleven in-frame vif gene mutants of HIV type 1 produced in non-permissive cells were examined for their replication potentials in various CD4-positive and -negative cell lines. Virus replication for each mutant was monitored by using several single- and multiple-cycle infectivity assays. Except for a mutant with wild-type phenotype, most mutants were severely defective for replication in all the cell lines as expected from the producer cell-dependent functioning of Vif so far reported. In contrast, two mutants, which have mutations in the hydrophilic or effector regions of Vif were found to have target cell-dependent replication potentials. These results demonstrate the presence of a novel category of the vif mutants important for elucidation of the Vif function.

Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells.

We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.

Apparent lack of trans-dominant negative effects of various vif mutants on the replication of HIV-1

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for virus growth in non-permissive cells such as H9. To elucidate the mechanism of action of the Vif protein, vif mutants, which show trans-dominant negative effects on the replication of HIV-1, would be useful tools. In this study, a new assay system to identify the mutants of this category was established. For this new system, various reporter clones carrying both mutant and authentic vif sequences were generated. By determining the growth ability of the viruses derived from the reporter constructs, the potential negative effect of the mutant vif sequence was readily and sensitively monitored. Ten vif mutant sequences tested were found not to exert the trans-dominant negative effect on the replication of HIV-1.

A Novel Interaction between FLICE-Associated Huge Protein (FLASH) and E2A Regulates Cell Proliferation and Cellular Senescence via Tumor Necrosis Factor (TNF)-Alpha-p21WAF1/CIP1 Axis

Dysregulation of the cell proliferation has been implicated in the pathophysiology of a number of diseases. Cellular senescence limits proliferation of cancer cells, preventing tumorigenesis and restricting tissue damage. However, the role of cellular senescence in proliferative nephritis has not been determined. The proliferative peak in experimental rat nephritis coincided with a peak in E2A expression in the glomeruli. Meanwhile, E12 (an E2A-encoded transcription factor) did not promote proliferation of Mesangial cells (MCs) by itself. We identified caspase-8-binding protein FLICE-associated huge protein (FLASH) as a novel E2A-binding partner by using a yeast two-hybrid screening. Knockdown of FLASH suppressed proliferation of MCs. This inhibitory effect was partially reversed by the knockdown of E2A. In addition, the knockdown of FLASH induced cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) expression, but did not affect p53 expression. Furthermore, overexpression of E12 and E47 induced p21, but not p53 in MCs, in the absence of FLASH. We also demonstrated that E2A and p21 expression at the peak of proliferation was followed by significant induction of FLASH in mesangial areas in rat proliferative glomerulonephritis. Moreover, we revealed that FLASH negatively regulates cellular senescence via the interaction with E12. We also demonstrated that FLASH is involved in the TNF-α-induced p21 expressions. These results suggest that the functional interaction of E2A and FLASH play an important role in cell proliferation and cellular senescence via regulation of p21 expression in experimental glomerulonephritis.

Novel Interplay Between Smad1 and Smad3 Phosphorylation via AGE Regulates the Progression of Diabetic Nephropathy

Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality compared with other causes of renal diseases. We previously found that Smad1 plays a critical role in the development of DN both in vitro and in vivo. However, functional interaction between Smad1 and Smad3 signaling in DN is unclear. Here, we addressed the molecular interplay between Smad1 and Smad3 signaling under a diabetic condition by using Smad3-knockout diabetic mice. Extracellular matrix (ECM) protein overexpression and Smad1 activation were observed in the glomeruli of db/db mice but were suppressed in the glomeruli of Smad3+/−; db/db mice. Smad3 activation enhanced the phosphorylation of Smad1 C-terminal domain but decreased the phosphorylation of linker domain, thus regulating Smad1 activation in advanced glycation end product-treated mesangial cells (MCs). However, forced phosphorylation of the Smad1 linker domain did not affect Smad3 activation in MCs. Phosphorylation of the Smad1 linker domain increased in Smad3+/−; db/db mice and probucol-treated db/db mice, which was consistent with the attenuation of ECM overproduction. These results indicate that Smad3 expression and activation or probucol treatment alters Smad1 phosphorylation, thus suggesting new molecular mechanisms underlying DN development and progression.