Andrew P. Rice

miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1

Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3′ untranslated region (3′UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression.

Regulation of Cyclin T1 and HIV-1 Replication by MicroRNAs in Resting CD4+ T Lymphocytes

ABSTRACT The replication of integrated human immunodeficiency virus type 1 (HIV-1) is dependent on the cellular cofactor cyclin T1, which binds the viral Tat protein and activates the RNA polymerase II transcription of the integrated provirus. The activation of resting CD4+ T cells upregulates cyclin T1 protein levels independently of an increase in cyclin T1 mRNA levels, suggesting a translational repression of cyclin T1 in resting CD4+ T cells. Hypothesizing that microRNAs (miRNAs) repress cyclin T1 translation in resting CD4+ T cells and that this inhibition is lifted upon cell activation, we used microarray expression analysis to identify miRNAs miR-27b, miR-29b, miR-150, and miR-223 as being significantly downregulated upon CD4+ T cell activation. The overexpression of these miRNAs decreased endogenous cyclin T1 protein levels, while treatment with the corresponding antagomiRs increased cyclin T1 protein levels. An miR-27b binding site within the cyclin T1 3′ untranslated region (3′UTR) was identified and confirmed to be functional after the mutation of key resides abrogated the ability of miR-27b to decrease the expression of a luciferase reporter upstream of the cyclin T1 3′UTR. Ago2 immunoprecipitation revealed an association with cyclin T1 mRNA that was decreased following treatment with miR-27b and miR-29b antagomiRs. Cells overexpressing miR-27b showed decreased viral gene expression levels of the HIV-1 reporter virus and a decreased replication of strain NL4.3; a partial rescue of viral transcription could be seen following the transfection of cyclin T1. These results implicate miR-27b as a novel regulator of cyclin T1 protein levels and HIV-1 replication, while miR-29b, miR-223, and miR-150 may regulate cyclin T1 indirectly.

Emerging Theme: Cellular PDZ Proteins as Common Targets of Pathogenic Viruses

ABSTRACT More than a decade ago, three viral oncoproteins, adenovirus type 9 E4-ORF1, human T-lymphotropic virus type 1 Tax, and high-risk human papillomavirus E6, were found to encode a related carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with a select group of cellular PDZ proteins. Recent studies have shown that many other viruses also encode PBM-containing proteins that bind to cellular PDZ proteins. Interestingly, these recently recognized viruses include not only some with oncogenic potential (hepatitis B virus, rhesus papillomavirus, cottontail rabbit papillomavirus) but also many without this potential (influenza virus, Dengue virus, tick-borne encephalitis virus, rabies virus, severe acute respiratory syndrome coronavirus, human immunodeficiency virus). Examination of the cellular PDZ proteins that are targets of viral PBMs reveals that the viral proteins often interact with the same or similar types of PDZ proteins, most notably Dlg1 and other members of the membrane-associated guanylate kinase protein family, as well as Scribble. In addition, cellular PDZ protein targets of viral PBMs commonly control tight junction formation, cell polarity establishment, and apoptosis. These findings reveal a new theme in virology wherein many different virus families encode proteins that bind and perturb the function of cellular PDZ proteins. The inhibition or perturbation of the function of cellular PDZ proteins appears to be a widely used strategy for viruses to enhance their replication, disseminate in the host, and transmit to new hosts.

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

The ESEV PDZ-Binding Motif of the Avian Influenza A Virus NS1 Protein Protects Infected Cells from Apoptosis by Directly Targeting Scribble

ABSTRACT The NS1 protein from influenza A viruses contains a four-amino-acid sequence at its carboxyl terminus that is termed the PDZ-binding motif (PBM). The NS1 PBM is predicted to bind to cellular PDZ proteins and functions as a virulence determinant in infected mice. ESEV is the consensus PBM sequence of avian influenza viruses, while RSKV is the consensus sequence of human viruses. Currently circulating highly pathogenic H5N1 influenza viruses encode an NS1 protein with the ESEV PBM. We identified cellular targets of the avian ESEV PBM and identified molecular mechanisms involved in its function. Using glutathione S-transferase (GST) pull-down assays, we found that the ESEV PBM enables NS1 to associate with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. Because Scribble possesses a proapoptotic activity, we investigated the interaction between NS1 and Scribble. The association between NS1 and Scribble is direct and requires the ESEV PBM and two Scribble PDZ domains. We constructed recombinant H3N2 viruses that encode an H6N6 avian virus NS1 protein with either an ESEV or mutant ESEA PBM, allowing an analysis of the ESEV PBM in infections in mammalian cells. The ESEV PBM enhanced viral replication up to 4-fold. In infected cells, NS1 with the ESEV PBM relocalized Scribble into cytoplasmic puncta concentrated in perinuclear regions and also protected cells from apoptosis. In addition, the latter effect was eliminated by small interfering RNA (siRNA)-mediated Scribble depletion. This study shows that one function of the avian ESEV PBM is to reduce apoptosis during infection through disruption of Scribbles proapoptotic function.

The Avian Influenza Virus NS1 ESEV PDZ Binding Motif Associates with Dlg1 and Scribble To Disrupt Cellular Tight Junctions

ABSTRACT The influenza A virus NS1 protein contains a conserved 4-amino-acid-residue PDZ-ligand binding motif (PBM) at the carboxyl terminus that can function as a virulence determinant by targeting cellular PDZ proteins. The NS1 proteins from avian and human viral isolates have consensus PBM sequences ESEV and RSKV, respectively. Currently circulating highly pathogenic H5N1 viruses contain the ESEV PBM which specifically associates with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. In this study, we found NS1 proteins from viral isolates that contain the PBM sequence RSKV, KSEV, or EPEV are unable to associate with these PDZ proteins. Other results showed that the ESEV PBM mediates an indirect association with PDZ protein, Lin7C, via an interaction with Dlg1. Infection with a virus that expresses a NS1 protein with the ESEV PBM results in colocalization of NS1, Scribble, and Dlg1 within perinuclear puncta and mislocalization of plasma membrane-associated Lin7C to the cytoplasm. Infection of polarized MDCK cells with the ESEV virus additionally results in functional disruption of the tight junction (TJ) as measured by altered localization of TJ markers ZO-1 and Occludin, decreased transepithelial electrical resistance, and increased fluorescein isothiocyanate (FITC)-inulin diffusion across the polarized cell monolayer. A similar effect on the TJ was observed in MDCK cells depleted for either Scribble or Dlg1 by small interfering RNA (siRNA). These findings indicate that ESEV PBM-mediated binding of NS1 to Scribble and Dlg1 functions to disrupt the cellular TJ and that this effect likely contributes to the severe disease associated with highly pathogenic H5N1 influenza A viruses.

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

Regulation of TAK / P-TEFb in CD4+ T Lymphocytes and Macrophages

HIV replication occurs principally in activated CD4+ T cells and macrophages. The HIV-1 Tat protein is essential for HIV replication and requires a cellular protein kinase activity termed TAK/P-TEFb, composed of CDK9 and cyclin T1, for its transactivation function. This article reviews recent work indicating that under some circumstances TAK/P-TEFb is likely to be limiting for HIV replication in CD4+ T cells and macrophages, and discusses mechanisms of regulation of the TAK/P-TEFb subunits in these cell types. In resting CD4+ T lymphocytes, TAK/P-TEFb function is low. Following lymphocyte activation, even under conditions of minimal activation in which activation markers and cellular proliferation are not induced, both CDK9 and cyclin T1 mRNA and protein levels are increased, leading to an induction of TAK/P-TEFb kinase activity that correlates with increased viral replication. In macrophages, regulation of TAK/P-TEFb involves mechanisms distinct from those in lymphocytes. In freshly isolated monocytes, CDK9 protein levels are high, while cyclin T1 protein levels are low to undetectable. Cyclin T1 protein expression is up-regulated during early macrophage differentiation by a mechanism that involves post-transcriptional regulation. Later during differentiation, cyclin T1 expression becomes shut off by a post-transcriptional mechanism, and this correlates with a decrease in Tat transactivation. Interestingly, cyclin T1 can be re-induced with lipopolysaccharide (LPS). These findings suggest that changes in cyclin T1 expression can influence HIV-1 replication levels in monocytes and macrophages. Important areas for future research on Tat and TAK/P-TEFb function are discussed.

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+ T Cells

ABSTRACT P-TEFb, a cellular kinase composed of Cyclin T1 and CDK9, is essential for processive HIV-1 transcription. P-TEFb activity is dependent on phosphorylation of Thr186 in the CDK9 T loop. In resting CD4+ T cells which are nonpermissive for HIV-1 replication, the levels of Cyclin T1 and T-loop-phosphorylated CDK9 are very low but increase significantly upon cellular activation. Little is known about how P-TEFb activity and expression are regulated in resting central memory CD4+ T cells, one of the main reservoirs of latent HIV-1. We used an in vitro primary cell model of HIV-1 latency to show that P-TEFb availability in resting memory CD4+ T cells is governed by the differential expression and phosphorylation of its subunits. This is in contrast to previous observations in dividing cells, where P-TEFb can be regulated by its sequestration in the 7SK RNP complex. We find that resting CD4+ T cells, whether naïve or memory and independent of their infection status, have low levels of Cyclin T1 and T-loop-phosphorylated CDK9, which increase upon activation. We also show that the decrease in Cyclin T1 protein upon the acquisition of a memory phenotype is in part due to proteasome-mediated proteolysis and likely also to posttranscriptional downregulation by miR-150. We also found that HEXIM1 levels are very low in ex vivo- and in vitro-generated resting memory CD4+ T cells, thus limiting the sequestration of P-TEFb in the 7SK RNP complex, indicating that this mechanism is unlikely to be a driver of viral latency in this cell type.

Phosphatase PPM1A Regulates Phosphorylation of Thr-186 in the Cdk9 T-loop

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to ∼50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.