Ann Florence B. Victoriano

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Latently Infected Cells by a Novel IκB Kinase Inhibitor

ABSTRACT In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-κB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-κB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-α) stimulates IκB kinase (IKK) complex, a critical regulator in the NF-κB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-α and IKK-β. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-α-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 μM, whereas its 50% cytotoxic concentration was about 15 μM. Western blot analysis revealed inhibition of IκBα phosphorylation, IκBα degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-κB activation. Furthermore, ACHP inhibited TNF-α-induced NF-κB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-α-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

Novel histone deacetylase inhibitor NCH-51 activates latent HIV-1 gene expression.

Pharmacological manipulations to purge human immunodeficiency virus (HIV) from latent reservoirs have been considered as an adjuvant therapeutic approach to highly‐active antiretroviral therapy for the eradication of HIV. Our novel histone deacetylase inhibitor NCH‐51 induced expression of latent HIV‐1 with minimal cytotoxicity. Using chromatin immunoprecipitation assays, we observed a reduction of HDAC1 occupancy, histone hyperacetylation and the recruitment of positive transcription factors at the HIV‐1 promoter in latently infected‐cells under the treatment with NCH‐51. Mutation studies of the long terminal repeat (LTR) revealed NCH‐51 mediated gene expression through the Sp1 sites. When Sp1 expression was knocked‐down by small interfering RNA, the NCH‐51‐mediated activation of a stably integrated HIV‐1 LTR was attenuated. Moreover, the Sp1 inhibitor mithramycin A abolished the effects of NCH‐51.

A Single-Nucleotide Synonymous Mutation in the gag Gene Controlling Human Immunodeficiency Virus Type 1 Virion Production

ABSTRACT Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.

Microbial Interaction of Periodontopathic Bacterium Porphyromonas gingivalis and HIV-Possible Causal Link of Periodontal Diseases to AIDS Progression-

A wide variety of infections, including bacteria, viruses, fungi and protozoa occur in the immunocompromised condition associated with human immunodeficiency virus type 1 (HIV-1) infection and acquired immunodeficiency syndrome (AIDS). Although these opportunistic infections are believed to arise as an effect of the immunodeficiency, these microbes sometimes promote the disease progression of HIV-1 infection by enhancing viral replication or modulating host immune responses. Here we review the experimental and clinical evidence supporting such causal relationships associated with periodontogenic bacteria. Periodontal disease, caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis (P. gingivalis) belonging to the phylum Bacteroidetes, is found worldwide and is one of the most prevalent microbial diseases of mankind. Emerging evidence implicates the involvement of P. gingivalis infection in the progression of HIV-1 infection. We demonstrate that P. gingivalis can induce HIV-1 reactivation via chromatin modification, and that the bacterial metabolite butyric acid produced in anaerobic conditions is responsible for this effect. These findings suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to AIDS progression. Furthermore, it would imply that prevention and early treatment of periodontitis involving P. gingivalis infection could effectively block further clinical progression of AIDS.

MicroRNAs in HIV-1 infection: an integration of viral and cellular interaction at the genomic level.

The microRNA pathways govern complex interactions of the host and virus at the transcripts level that regulate cellular responses, viral replication and viral pathogenesis. As a group of single-stranded short non-coding ribonucleotides (ncRNAs), the microRNAs complement their messenger RNA (mRNA) targets to effect post-transcriptional or translational gene silencing. Previous studies showed the ability of human immunodeficiency virus 1 (HIV-1) to encode microRNAs which modify cellular defence mechanisms thus creating an environment favorable for viral invasion and replication. In corollary, cellular microRNAs were linked to the alteration of HIV-1 infection at different stages of replication and latency. As evidences further establish the regulatory involvement of both cellular and viral microRNA in HIV-1-host interactions, there is a necessity to organize this information. This paper would present current and emerging knowledge on these multi-dimensional interactions that may facilitate the design of microRNAs as effective antiretroviral reagents.

Cyclin T1 stabilizes expression levels of HIV‐1 Tat in cells

Transcription from HIV‐1 proviral DNA is a rate‐determining step for HIV‐1 replication. Interaction between the cyclin T1 (CycT1) subunit of positive transcription elongation factor b (P‐TEFb) and the Tat transactivator protein of HIV‐1 is crucial for viral transcription. CycT1 also interacts directly with the transactivation‐responsive element (TAR) located on the 5′end of viral mRNA, as well as with Tat through the Tat–TAR recognition motif (TRM). These molecular interactions represent a critical step for stimulation of HIV transcription. Thus, Tat and CycT1 are considered to be feasible targets for the development of novel anti‐HIV therapies. In this study, we demonstrate that CycT1 is positively involved in the Tat protein stability. Selective degradation of CycT1 by small interfering RNA (siRNA) culminated in proteasome‐mediated degradation of Tat and eventual inhibition of HIV‐1 gene expression. We noted that the siRNA‐mediated knockdown of CycT1 could inhibit HIV‐1 transcription without affecting cell viability and Tat mRNA levels. These findings clearly indicate that CycT1 is a feasible therapeutic target, and inactivation or depletion of CycT1 should effectively inhibit HIV replication by destabilizing Tat and suppressing Tat‐mediated HIV transcription.

Functional characterization of human cyclin T1 N-terminal region for human immunodeficiency virus-1 Tat transcriptional activation.

Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat-TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)-Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.

Interaction between Endogenous Bacterial Flora and Latent HIV Infection

ABSTRACT Human commensal bacteria do not normally cause any diseases. However, in certain pathological conditions, they exhibit a number of curious behaviors. In HIV infection, these bacteria exhibit bidirectional relationships: whereas they cause opportunistic infections based on immunological deterioration, they also augment HIV replication, in particular, viral replication from latently infected cells, which is attributable to the effect of butyric acid produced by certain anaerobic bacteria by modifying the state of chromatin. Here, we review recent evidence supporting the contributory role of such endogenous microbes in disrupting HIV latency and its potential link to the clinical progression of AIDS.