Regulation of cyclin T1 expression and function by an alternative splice variant that skips exon 7 and contains a premature termination codon.

Cyclin T1 (CCNT1), a gene containing nine exons, forms the positive transcription elongation factor b (P-TEFb) complex and regulates a wide variety of biological processes including transcription. We discovered a novel splice variant of CCNT1 that lacks exon 7 (dE7). RT-PCR analysis revealed that the dE7 transcript was detected in almost all tissues examined. The dE7/FL transcript ratio was high in quiescent peripheral blood mononuclear cells (PBMC) and in tissues poor in cell division; however, it was low in activated PBMC and in tissues with high cell proliferative potential. These results suggest that exon 7 skipping is linked to cell cycle progression. Increasing the dE7/FL transcript ratio resulted in the reduction of CCNT1 protein levels, indicating that the expression of CCNT1 protein is controlled by exon skipping. Exon 7 skipping yields a +1 frameshift at exon 8, which generates a premature termination codon (PTC). The dE7 transcript levels increased when cells were treated with the protein synthesis inhibitor cycloheximide (CHX) or a kinase inhibitor wortmannin (WORT), whilst the FL transcript levels were unchanged, suggesting that the dE7 transcript is a target of nonsense-mediated decay (NMD). Importantly, reduction of dE7 transcript by WORT correlated well with the decrement of CCNT1 protein expression. The dE7 transcript would produce an approximately 23kDa protein that covers approximately 70% of the cyclin box. The ectopically expressed dE7 protein physically interacted with CDK9 and competed with FL CCNT1 for CDK9, thus should act dominant-negatively on FL CCNT1. The replication of human immunodeficiency virus type 1 (HIV-1), heavily dependent on the CCNT1 function, was inhibited by dE7 protein through the attenuation of Tat/long terminal repeat (LTR)-driven transcription. Taken together, these results suggest that dE7 is a novel splice variant that regulates the expression and function of CCNT1.

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.

Ligand-independent higher-order multimerization of CXCR4, a G-protein-coupled chemokine receptor involved in targeted metastasis.

CXCR4, a G-protein-coupled receptor of CXCL12/stromal cell-derived factor-1alpha, mediates a wide range of physiological and pathological processes, including the targeted metastasis of cancer cells. CXCR4 has been shown to homo-oligomerize in several experimental systems. However, it remains unclear with which domains CXCR4 interacts homotypically, and whether it dimerizes or forms a higher-order complex. To address these issues, we used bioluminescent resonance energy transfer and bimolecular fluorescence complementation analyses to measure the homotypic interactions of CXCR4 in living cells. Both assays indicated that CXCR4 interacts homotypically, which is consistent with previous studies. By studying CXCR4 mutants lacking various domains, we found that multiple transmembrane domains probably serve as potential molecular interaction surfaces for oligomerization. The relative contribution of the amino- or carboxy-termini to oligomerization was small. To differentiate between a dimer and a multimer consisting of more than two molecules, bioluminescent resonance energy transfer-bimolecular fluorescence complementation analysis was conducted. It revealed that CXCR4 engages in higher-order oligomerization in a ligand-independent fashion. This is the first report providing direct experimental evidence for the higher-order multimerization of CXCR4 in vivo. We hypothesize that CXCR4 distributes to the cell surface as a multimer, in order to effectively sense, with increased avidity, the chemotaxis-inducing ligand in the microenvironment. Studying the structure and function of the oligomeric state of CXCR4 may lead us to develop novel CXCR4 inhibitors that disassemble the molecular cluster of CXCR4.

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.

Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-delta1 pleckstrin homology domain results in infectious pseudovirion production.

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-delta1 pleckstrin homology (PH) domain. PH-Gag-Pol PM targeting and viral production efficiencies were improved compared with Gag-Pol, consistent with the estimated increases in Gag-PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH-Gag-Pol and Gag-Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH-Gag-Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.

Cyclin K/CPR4 inhibits primate lentiviral replication by inactivating Tat/positive transcription elongation factor b-dependent long terminal repeat transcription.

The positive transcription elongation factor b complexes comprise CDK9 and a C-type cyclin, required for the efficient expression of both eukaryotic and primate lentivirus-encoded genes. Cyclin K/CPR4 is the least studied of the positive transcription elongation factor b-forming cyclins. Here, we demonstrate that cyclin K/CPR4-containing positive transcription elongation factor b complexes are unresponsive to Tat and HEXIM1-mediated inactivation. Enhancing expression of cyclin K/CPR4 inhibited the human and simian immunodeficiency viral replication. These data indicate that cyclin K/CPR4 functions as a natural inhibitor of primate lentiviruses.

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.

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-1alpha)/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-1alpha. 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 Ser(344) Glu(345) Ser(346) are essential for efficient steady-state internalization of CXCR4. Mutating Glu(345) to Asp did not disrupt internalization, suggesting that the steady-state internalization motif is S(E/D)S. When responses to SDF-1alpha 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-1alpha. 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.

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 sparsomycin's 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.

The interaction of HIV-1 with the host factors.

Human immunodeficiency virus type 1 (HIV-1) is a causative agent of acquired immunodeficiency syndrome (AIDS) in humans. In the last decade, the functions of HIV-1-encoded genes have been intensively studied. These studies have contributed to the development of the effective anti-AIDS drugs directing against the HIV-1-encoded enzymes, namely reverse transcriptase and protease. However, even the combination of these drugs is not sufficient enough to stop the progression of AIDS partly due to the emergence of drug-resistant HIV-1 mutants as well as the severe side effects. Understanding the molecular mechanisms by which cellular factors support the efficient replication of HIV-1 should contribute to develop means to control the progression of AIDS. This field is now expanding rapidly. Here we review the host factors involved in the replication of HIV-1 and highlight some findings that have a substantial impact on the retroviral research.