Regulation Of HIV-1 Replication Research Articles

Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs.

Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.

Regulation of CDK9 Activity by Phosphorylation and Dephosphorylation

HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1.

Identification of HIV-1 sequences targeted by the miRNA Induced silencing complex (miRISC) using Argonaute 2 cross-linking and immunoprecipitation (HITS-CLIP)

Background The RNA interference pathway (RNAi) regulates nearly 50 percent of human transcripts at a post-transcriptional level. The effector complex of the pathway is the microRNA-induced silencing complex (miRISC) composed of one of the four Argonaute proteins (Ago1 to 4) associated with a miRNA. Once loaded into the miRISC, these small non-coding RNA guide the complex to specific mRNA sequences and the binding of the Ago protein to the transcripts can result either in its degradation or in translational repression. Several studies have shown that the miRNA pathway can also participate in the regulation of HIV-1 replication and latency. However, the molecular mechanisms involved remain to be explored. Using a HITS-CLIP (high throughput sequencing of RNA isolated by cross-linking and immunoprecipitation) approach, we identified viral RNA sequences targeted by the miRISC.

Iron depletion by iron chelators or ferroportin inhibits HIV-1 through the induction of HIF1α, p21 and IKBα and the inhibition of CDK9 and CDK2

Background Intracellular iron and oxygen levels regulate HIV-1 replication by affecting several steps in the HIV-1 life cycle including transcription [1]. Low oxygen levels and reduced cellular iron are inhibitory as CDK9/cyclin T1 and CDK2/ cyclin E activities are reduced and HIV-1 transcription is inhibited. The alpha subunit of hypoxia-induced factor 1 (HIF1a) is stabilized under hypoxia and in the conditions of low cellular iron. Iron depletion by iron chelators or through the expression of ferroportin, an iron export protein, inhibits CDK2 and CDK9 activities and blocks HIV-1 transcription [1]. As neither CDK2 nor CDK9 requires iron for their enzymatic activity, an apparent upstream regulation is involved. Also, induction of heme oxygenase-1 (HO-1) by hemin was shown to inhibit HIV-1 [1] although the mechanism of the inhibition was not clarified. Here we analyzed the effect of iron chelators on HIV-1 transcription and replication and also analyzed the effect of heme, a condition present in sickle cell disease that may protect against HIV-1 infection [2].

Some mechanisms of FLIP expression in inhibition of HIV‐1 replication in Jurkat cells, CD4+ T cells and PBMCs

HIV-1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV-1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV-1 replication are critical to understanding the mechanisms of HIV-1 replicative cycle. Using the susceptible Jurkat cell line, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase-8, in HIV-1 production. Full length cellular FLIP (cFLIP) inhibited HIV-1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV-1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV-1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas-mediated apoptotic pathway. The inactivated FADD also inhibited HIV-1 replication.

Kuan-Teh Jeang

Kuan-Teh Jeang

Role of Cellular Iron and Oxygen in the Regulation of HIV-1 Infection

Despite efficient antiretroviral therapy, eradication of HIV-1 infection is challenging and requires novel biological insights and therapeutic strategies. Among other physiological and environmental factors, intracellular iron greatly affects HIV-1 replication. Higher iron stores were shown to be associated with faster progression of HIV-1 infection and to inversely correlate with the survival of HIV-1 infected patients. Iron is required for several steps in the HIV-1 life cycle, including reverse transcription, HIV-1 gene expression and capsid assembly. Here, the authors present a comprehensive review of the molecular mechanisms involved in iron- and oxygen-mediated regulation of HIV-1 replication. We also propose key intracellular pathways that may be involved in regulating HIV-1 replication, via protein kinase complexes, CDK9/cyclin T1 and CDK 2/cyclin E, protein phosphatase-1 and other host factors.

Genome-wide association analysis in host characteristics of progression to high-grade cervical intraepithelial neoplasia for women with human papilloma virus infection and normal cytology.

5109 Background: Although it is well accepted that persistent human papillomavirus (HPV) is necessary for carcinogenesis of cervical neoplasms, the molecular mechanism for progression is unclear. HPV testing is widely used for cervical cancer screening. The hazard ratio of developing cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) in baseline HPV-positive/normal cytology women is 30-50 fold as compared with HPV-negative/normal cytoloy women. HPV positivity would cause substantial anxiety. It is important to identify a prognostic profile for predicting progression. Methods: We collected blood samples from women aged ≥ 30 years in a population-based nested cohort study enrolling women with HPV infection (n = 871) or HPV-negative (n = 902) with normal cytology in 2004-2009 for prospective follow-up. To identify the host genetic characteristics associated with cervical carcinogenesis, a genome-wide association study (analyzing 530,194 SNPs) was conducted in 23 cases who developed CIN2+ and 62 viral type- and age-matched controls who were HPV-positive at baseline without developing CIN throughout the follow-up period. Results: One SNP with significant P values (rs16969682; P=4.58 x 10-5, OR=5.9, 95% CI=2.52-13.96) located in SEC14-like 1 (SEC14L1) gene localized to chromosome 17 was identified with association between progression to CIN2+ and controls without CIN throughout follow-up. SEC14L1 belongs to the widely-expressed SEC14-superfamily. This superfamily consists of > 500 members that are involved in biological functions including membrane trafficking and phospolipid metabolism. Furthermore, using T-cell based cDNA screening, SEC14L-1a is identified as a regulator of HIV-1 replication. Conclusions: The potential role of SEC14L1 in host-viral interaction will be further elucidated. Additional statistically significant SNPs including rs16969682 will be validated on cases with CIN2+ (n = 250) and normal controls (normal cytology/HPV-negative at baseline without acquisition of HPV or abnormal cytology/histology during follow-up, n = 500).

Protein kinase C-delta regulates HIV-1 replication at an early post-entry step in macrophages

BackgroundMacrophages, which are CD4 and CCR5 positive, can sustain HIV-1 replication for long periods of time. Thus, these cells play critical roles in the transmission, dissemination and persistence of viral infection. Of note, current antiviral therapies do not target macrophages efficiently. Previously, it was demonstrated that interactions between CCR5 and gp120 stimulate PKC. However, the PKC isozymes involved were not identified.ResultsIn this study, we identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. Indeed, PKC-delta was stimulated following the interaction between the virus and its target cell. Moreover, inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. However, this inhibition did not have significant effects on receptor and co-receptor expression or fusion. Additionally, it did not affect the formation of the early reverse transcription product containing R/U5 sequences, but did inhibit the synthesis of subsequent cDNAs. Importantly, the inhibition of PKC-delta altered the redistribution of actin, a cellular cofactor whose requirement for the completion of reverse transcription was previously established. It also prevented the association of the reverse transcription complex with the cytoskeleton.ConclusionThis work highlights the importance of PKC-delta during early steps of the replicative cycle of HIV-1 in human macrophages.

Mov10, an APOBEC3G-interacting RNA-binding protein, inhibits HIV-1 infection

Background Identification of cellular factors that negatively or positively regulate HIV-1 replication is essential to understand HIV-1 pathogenesis for development of novel antiviral treatments. The human cytidine deaminase APOBEC3G (A3G) is an intrinsic antiviral factor important for the host-mediated defence against HIV-1. We and others recently identified a panel of A3G-interacting RNA-binding proteins (RBPs) that focused our search for novel HIV-1 cofactors and restriction factors onto these proteins. Mov10, a superfamily-1 putative RNA helicase, interacts with A3G in an RNA-dependent manner. Mov10 associates with the RNA-induced silencing complex pathway and localises to mRNA processing bodies (PBs). Furthermore, orthologs of Mov10 in Arabidopsis thaliana and Drosophila melanogaster are necessary for antiviral small RNA-mediated silencing. Thus, we investigated whether Mov10 restricts HIV-1 replication.

Association of Vpu with hepatitis C virus NS3/4A stimulates transcription of type 1 human immunodeficiency virus

Type 1 human immunodeficiency virus (HIV-1) and hepatitis C virus (HCV) are deadly bloodborne-transmitting pathogens. Due to sharing the routes of transmission, co-infection of HIV-1 and HCV is common with a high rate. Co-infection of HCV affects morbidity and mortality of patients with AIDS and impairs their tolerance to antiretroviral therapy. In this study, the roles of HCV proteins in the regulation of HIV-1 replication and the molecular mechanism involved in such regulation were investigated. We demonstrated that HCV NS3 protein stimulated HIV-1 LTR transcription and that HIV-1 Vpu protein was required for the activation of HIV-1 transcription regulated by HCV NS3/4A complex. Further study revealed that Vpu mediated ubiquitination-associated degradation of NS4A, detached NS3/4A complex and release NS3 for nuclear translocation. Since both degradation of NS4A and activation of HIV-1 LTR were closely correlated and mediated by Vpu, we proposed that Vpu impairs the stability of NS4A and releases NS3 from NS3/4A complex for the stimulation of HIV-1 transcription. This study enriched our understanding on HIV-1/HCV co-infection and provided new insights in molecular mechanism involved in the co-infection of the two viruses.

HIV-1 Replication and Latency Are Regulated by Translational Control of Cyclin T1

Human immunodeficiency virus (HIV) exploits cellular proteins during its replicative cycle and latent infection. The positive transcription elongation factor b (P-TEFb) is a key cellular transcription factor critical for these viral processes and is a drug target. During viral replication, P-TEFb is recruited via interactions of its cyclin T1 subunit with the HIV Tat (transactivator of transcription) protein and TAR (transactivation response) element. Through RNA silencing and over-expression experiments, we discovered that nuclear factor 90 (NF90), a cellular RNA binding protein, regulates P-TEFb expression. NF90 depletion reduced cyclin T1 protein levels by inhibiting translation initiation. Regulation was mediated by the 3′ untranslated region of cyclin T1 mRNA independently of microRNAs. Cyclin T1 induction is involved in the escape of HIV-1 from latency. We show that the activation of viral replication by phorbol ester in latently infected monocytic cells requires the posttranscriptional induction of NF90 and cyclin T1, implicating NF90 in protein kinase C signaling pathways. This investigation reveals a novel mechanism of cyclin T1 regulation and establishes NF90 as a regulator of HIV-1 replication during both productive infection and induction from latency.

Insights into Cellular Factors That Regulate HIV-1 Replication in Human Cells

Retroviruses integrate into the host cell's chromosome. Accordingly, many aspects of the life cycle of retroviruses like HIV-1 are intimately linked to the functions of cellular proteins and RNAs. In this review, we discuss in brief recent genomewide screens for the identification of cellular proteins that assist HIV-1 replication in human cells. We also review findings for other cellular moieties that help or restrict the viral life cycle.

T cell-based functional cDNA library screening identified SEC14-like 1a carboxy-terminal domain as a negative regulator of human immunodeficiency virus replication

Genome-wide screening of host factors that regulate HIV-1 replication has been attempted using numerous experimental approaches. However, there has been limited success using T cell-based cDNA library screening to identify genes that regulate HIV-1 replication. We have established a genetic screening strategy using the human T cell line MT-4 and a replication-competent HIV-1. With this system, we identified the C-terminal domain (CTD) of SEC14-like 1a (SEC14L1a) as a novel inhibitor of HIV-1 replication. Our T cell-based cDNA screening system provides an alternative tool for identifying novel regulators of HIV-1 replication.

Host hindrance to HIV-1 replication in monocytes and macrophages

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. HIV-1 can replicate in blood monocytes, although only a minor proportion of circulating monocytes harbor viral DNA. Resident macrophages in tissues can be infected and function as viral reservoirs. However, their susceptibility to infection, and their capacity to actively replicate the virus, varies greatly depending on the tissue localization and cytokine environment. The susceptibility of monocytes to HIV-1 infection in vitro depends on their differentiation status. Monocytes are refractory to infection and become permissive upon differentiation into macrophages. In addition, the capacity of monocyte-derived macrophages to sustain viral replication varies between individuals. Host determinants regulate HIV-1 replication in monocytes and macrophages, limiting several steps of the viral life-cycle, from viral entry to virus release. Some host factors responsible for HIV-1 restriction are shared with T lymphocytes, but several anti-viral mechanisms are specific to either monocytes or macrophages. Whilst a number of these mechanisms have been identified in monocytes or in monocyte-derived macrophages in vitro, some of them have also been implicated in the regulation of HIV-1 infection in vivo, in particular in the brain and the lung where macrophages are the main cell type infected by HIV-1. This review focuses on cellular factors that have been reported to interfere with HIV-1 infection in monocytes and macrophages, and examines the evidences supporting their role in vivo, highlighting unique aspects of HIV-1 restriction in these two cell types.

Novel HIV-1 therapeutics through targeting altered host cell pathways

The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse transcriptase, viral protease or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host's proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be used to better design therapeutics against HIV-1.

HIV-1 transactivator protein induction of suppressor of cytokine signaling-2 contributes to dysregulation of IFNγ signaling

AbstractHIV infection remains a worldwide threat. HIV-1 transactivator protein Tat is one of the retroviral proteins identified as a key immunomodulator in AIDS pathogenesis. Although the primary function of Tat is to regulate HIV-1 replication in the infected cell, it also dysregulates cytokine production resulting in perturbation of the host immune response and enhancement of the retrovirus survival. Because interferon-γ (IFNγ) is a pleiotropic cytokine with potent antiviral and immunoregulatory effects, we investigated whether Tat interferes with the IFNγ signal transduction in primary monocytes. We demonstrated that Tat impaired the IFNγ-receptor signaling pathway at the level of STAT1 activation, possibly via Tat-dependent induction of suppressor of cytokine signaling-2 (SOCS-2) activity. We delineated the inhibitory role of SOCS-2 in IFNγ signaling pathway by overexpression of exogenous SOCS-2 in HEK293 cell. The results showed that SOCS-2 suppressed the IFNγ-activated STAT1 phosphorylation and consequent IFNγ-regulated transcription of specific genes. To confirm the role of SOCS2 in the Tat-induced process, we demonstrated that SOCS-2 siRNA in human blood monocytes abrogated the Tat-dependent inhibition of IFNγ signaling. Our data suggested a possible mechanism implicating the role of SOCS-2 in mediating HIV-1–induced immune evasion and dysregulation of IFNγ signaling in primary human monocytes.

Tumor Necrosis Factor-alpha Levels in HIV-1 Seropositive Injecting Drug Users

TNF-alpha is a highly pleiotropic cytokine and plays an important role in regulating HIV-1 replication. It may compromise the integrity of the blood-brain-barrier and, thus, may contribute to the neurotoxicity of HIV-1-infection. Both intravenous drug abuse (IDU) and HIV infection can increase TNF-alpha activity, but little information is available on the effects of a combination of these factors on TNF-alpha. We investigated plasma TNF-alpha levels and mRNA in the peripheral monocytes of 166 men and women in three groups: HIV-1-positive IDUs, HIV-1-negative IDUs, and HIV-negative non-IDU control participants. HIV-1-positive IDUs had higher TNF-alpha levels than HIV-1-negative IDUs who, in turn, had higher levels than controls. TNF-alpha mRNA expression in peripheral monocytes was significantly increased in both HIV-1-positive and negative IDUs compared to controls. These findings show that the effects of HIV infection and intravenous drug use may be additive in increasing TNF-alpha levels. Given the multiple effects of TNF-alpha in HIV infection, additional investigation of its role is needed.

Suppression of HIV-1 replication by microRNA effectors

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.

Pathway Analysis in HEK 293T Cells Overexpressing HIV-1 Tat and Nucleocapsid

The human immunodeficiency virus (HIV)-1 protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA microarrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the microarray data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.