Jun Komano

Epstein-Barr virus provides a survival factor to Burkitt's lymphomas

Epstein-Barr virus (EBV) has been causally associated with at least five human malignancies. The exact contributions made by EBV to these cancers remain unknown. We demonstrate that one viral protein found in all EBV-associated malignancies, Epstein-Barr nuclear antigen 1 (EBNA-1), is required for survival of one of these cancers, EBV-positive Burkitts lymphoma. Inhibition of EBNA-1 decreases survival of these tumor cells by inducing apoptosis. Expression of EBNA-1 in uninfected cells also can inhibit apoptosis induced by expression of p53 in the absence of the EBV genome. Our findings demonstrate that EBNA-1 is critical for the continued survival of EBV-associated Burkitts lymphoma, and, by extension, for the other B cell tumors with which EBV is associated. Efficient inhibitors of EBNA-1s functions would likely prove useful in the therapy of EBV-associated malignancies.

Role of the Specific Amino Acid Sequence of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 in Membrane Fusion

ABSTRACT Fusion between cell and virus membranes mediated by gp41 initiates the life cycle of human immunodeficiency virus type 1. In contrast to the many studies that have elucidated the structure-function relationship of the ectodomain, the study of the membrane-spanning domain (MSD) has been rather limited. In particular, the role that the MSDs specific amino acid sequences may have in membrane fusion as well as other gp41 functions is not well understood. The MSD of gp41 contains well-conserved glycine residues that form the GXXXG motif (G, glycine; X, other amino acid residues), a motif often found at the helix-helix interface of membrane spanning α-helices. Here we examined the role that the specific amino acid sequence of the gp41 MSD has in gp41 function, particularly in membrane fusion, by making two types of MSD mutants: (i) glycine substitution mutants in which glycine residues of the MSD were mutated to alanine or leucine residues, and (ii) replacement mutants in which the entire MSD was replaced with one derived from glycophorin A or from vesicular stomatitis virus G. The substitution of glycines did not affect gp41 function. MSD-replacement mutants, however, showed severely impaired fusion activity. The assay using the Env expression vector revealed defects in membrane fusion after CD4 binding steps in the MSD-replacement mutants. In addition, the change in Env processing was noted for MSD-replacement mutants. These results suggest that the MSD of gp41 has a relatively wide but not unlimited tolerance for mutations and plays a critical role in membrane fusion as well as in other steps of Env biogenesis.

SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus–host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.

A CD63 Mutant Inhibits T-cell Tropic Human Immunodeficiency Virus Type 1 Entry by Disrupting CXCR4 Trafficking to the Plasma Membrane

We have discovered that an N‐terminal deletion mutant of a membrane protein, CD63, (CD63ΔN) blocks entry of CXCR4‐using, T‐cell tropic human immunodeficiency virus type 1 (X4 HIV‐1) by suppressing CXCR4 surface expression. This suppression was observed for CXCR4 but not for CD4, CCR5, CD25, CD71 or other tetraspanin proteins. The suppression of CXCR4 expression on the plasma membrane appeared to be caused by mislocalization of CXCR4 and exclusive transportation of CXCR4 toward intracellular organelles, mainly late endosomes/lysosomes. Our data suggest that CXCR4 trafficking can be modified in terms of its recruitment to the plasma membrane without enhancing the degradation or arresting vesicular transport of CXCR4.

Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4

The C‐terminal cytoplasmic domain of the metastatic potentiator CXCR4 regulates its function and spatiotemporal expression. However, little is known about the mechanism underlying constitutive internalization of CXCR4 compared to internalization mediated by its ligand, stromal cell‐derived factor‐1 alpha (SDF‐1α)/CXCL12. We established a system to analyze the role of the CXCR4 cytoplasmic tail in steady‐state internalization using the NP2 cell line, which lacks endogenous CXCR4 and SDF‐1α. Deleting more than six amino acids from the C‐terminus dramatically reduced constitutive internalization of CXCR4. Alanine substitution mutations revealed that three of those amino acids Ser344 Glu345 Ser346 are essential for efficient steady‐state internalization of CXCR4. Mutating Glu345 to Asp did not disrupt internalization, suggesting that the steady‐state internalization motif is S(E/D)S. When responses to SDF‐1α were tested, cells expressing CXCR4 mutants lacking the C‐terminal 10, 14, 22, 31 or 44 amino acids did not show downregulation of cell surface CXCR4 or the cell migration induced by SDF‐1α. Interestingly, however, we identified two mutants, one with E344A mutation and the other lacking the C‐terminal 17 amino acids, that were defective in constitutive internalization but competent in ligand‐promoted internalization and cell migration. These data demonstrate that ligand‐dependent and ‐independent internalization is genetically separable and that, between amino acids 336 and 342, there is a negative regulatory element for ligand‐promoted internalization. Potential involvement of this novel motif in cancer metastasis and other CXCR4‐associated disorders such as warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome is discussed. (Cancer Sci 2007; 98: 373–379)

Derivatives of 5-nitro-furan-2-carboxylic acid carbamoylmethyl ester inhibit RNase H activity associated with HIV-1 reverse transcriptase.

The RNase H activity associated with human immunodeficiency virus type 1 (HIV-1) is an attractive target for an antiretroviral drug development. We screened 20000 small-molecular-weight compounds for RNase H inhibitors and identified a novel RNase H-inhibiting structure characterized by a 5-nitro-furan-2-carboxylic acid carbamoylmethyl ester (NACME) moiety. Two NACME derivatives, 5-nitro-furan-2-carboxylic acid adamantan-1-carbamoylmethyl ester (compound 1) and 5-nitro-furan-2-carboxylic acid [[4-(4-bromo-phenyl)-thiazol-2-yl]-(tetrahydro-furan-2-ylmethyl)-carbamoyl]-methyl ester (compound 2), effectively blocked HIV-1 and MLV RT-associated RNase H activities with IC(50)s of 3-30 microM but had little effect on bacterial RNase H activity in vitro. Additionally, 20-25 microM compound 2 effectively inhibited HIV-1 replication. An in silico docking simulation indicated that the conserved His539 residue, and two metal ions in the RNase H catalytic center are involved in RNase H inhibition by NACME derivatives. Taken together, these data suggest that NACME derivatives may be potent lead compounds for development of a novel class of antiretroviral drugs.

Inhibiting lentiviral replication by HEXIM1, a cellular negative regulator of the CDK9/cyclin T complex.

Objective:Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. Methods:HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. Results:HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. Conclusion:These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.

Role of bcl-2 in Epstein-Barr Virus-Induced Malignant Conversion of Burkitt's Lymphoma Cell Line Akata

ABSTRACT We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitts lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.

Identification of the P-TEFb complex-interacting domain of Brd4 as an inhibitor of HIV-1 replication by functional cDNA library screening in MT-4 cells

We conducted a phenotypic cDNA screening using a T cell line‐based assay to identify human genes that render cells resistant to human immunodeficiency virus type 1 (HIV‐1). We isolated potential HIV‐1 resistance genes, including the carboxy terminal domain (CTD) of bromodomain‐containing protein 4 (Brd4). Expression of GFP‐Brd4‐CTD was tolerated in MT‐4 and Jurkat cells in which HIV‐1 replication was markedly inhibited. We provide direct experimental data demonstrating that Brd4‐CTD serves as a specific inhibitor of HIV‐1 replication in T cells. Our method is a powerful tool for the identification of host factors that regulate HIV‐1 replication in T cells.

Rapid Propagation of Low-Fitness Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 by a Streptococcal Metabolite Sparsomycin

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycins ability to increase the –1 frameshift efficiency required to produce the Gag—Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.