Regulation Of Viral Transcription Research Articles

HSV-1 miR-H6 Inhibits HSV-1 Replication and IL-6 Expression in Human Corneal Epithelial Cells In Vitro

HSV-1 infection in the cornea could lead to blindness. The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. It has been previously demonstrated that miR-H6 encoded from HSV-1 genome targets ICP4 to help maintain latency. In this study, synthesized miR-H6 mimics were transfected into HSV-1-infected human cornea epithelial (HCE) cells. The inhibition of HSV-1 replication and viral ICP4 expression in miR-H6-transfected HCE was confirmed by plaque assay, immunofluorescence, and Western blot. Compared to nontransfection or mock, miR-H6 produced a low-titer HSV-1 and weak ICP4 expression. In addition, miR-H6 can decrease the interleukin 6 released into the medium, which was determined by ELISA. Taken together, the data suggests that miR-H6 targeting of ICP4 inhibits HSV-1 productive infection and decreases interleukin 6 production in HCE, and this may provide an approach to prevent HSV-1 lytic infection and inhibit corneal inflammation.

Identification of target genes for the CDK subunits of the Mediator complex

Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate the in vivo transcriptional activities of the complexes, we used a luciferase assay in combined with siRNA-mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16-dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context-specific transcriptional regulator.

Cdc20 mediates D-box-dependent degradation of Sp100

Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.

A Theoretical Model for the Dynamic Structure of Hepatitis B Nucleocapsid

The genomic material of hepatitis B virus (HBV) is confined within a fenestrated nucleocapsid consisting of 240 identical copies of the capsid protein, which has a rigid core and a positively charged and highly flexible C-terminal domain (CTD). Although previous mutagenesis studies have demonstrated the importance of the CTD in viral RNA packaging and reverse transcription, the microscopic structure of the CTD and its interaction with encapsidated nucleic acids at various stages of viral maturation remain poorly understood. Here, we present a theoretical analysis of the radial distributions of the CTD chains and nucleic acids in the hepatitis B virus nucleocapsid at the beginning and final stages of viral reverse transcription based on classical density functional theory and a coarse-gained model for the pertinent biomolecules. We find that a significant portion of the CTD is exposed at the surface of the RNA-containing immature nucleocapsid and that the CTD is mostly confined within the DNA-containing mature nucleocapsid. Large accumulation of cations is predicted inside both immature and mature nucleocapsids. The theoretical results provide new insights into the molecular mechanism of CTD regulation of viral reverse transcription and nucleocapsid trafficking during various stages of the viral replication processes.

Identification and Analysis of Papillomavirus E2 Protein Binding Sites in the Human Genome

Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs-E2 binding sites (E2BS)-in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functionality of E2BSs in the human genome. Our computational analysis indicates that most E2BSs in the human genome are found in repetitive DNA regions and have G/C-rich spacer sequences. Using a chromatin immunoprecipitation approach, we show that human papillomavirus type 11 (HPV11) E2 interacts with a subset of cellular E2BSs located in active chromatin regions. Two E2 activities, sequence-specific DNA binding and interaction with cellular Brd4 protein, are important for E2 binding to consensus sites. E2 binding to cellular E2BSs has a moderate or no effect on cellular transcription. We suggest that the preference of HPV E2 proteins for E2BSs with A/T-rich spacers, which are present in the viral genomes and underrepresented in the human genome, ensures E2 binding to specific binding sites in the virus genome and may help to prevent extensive and possibly detrimental changes in cellular transcription in response to the viral protein.

Identification of BPR3P0128 as an Inhibitor of Cap-Snatching Activities of Influenza Virus

The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin-Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of cap-dependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5' viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.

HTLV-1 bZIP factor enhances TGF-β signaling through p300 coactivator

AbstractHuman T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-β (TGF-β)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-β signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-β response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-β. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-β signaling. Our results suggest that HBZ, by enhancing TGF-β signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence.

Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4nM in primary macrophages and 0.5nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity.

HSV-1 stimulation-related protein HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2

The protein encoded by HSRG1 (HSV-1 stimulation-related gene 1) is a virally induced protein expressed in HSV-1-infected cells. We have already reported that HSRG1 is capable of interacting with transcriptional regulator proteins. To further analyze the effects of HSRG1 on the regulation of viral gene transcription, we expressed the HSRG1 protein in transfected cells and found that it postpones the proliferation of HSV-1. CAT (chloramphenicol acetyltransferase) assays also revealed that HSRG1 reduces transcription from HSV-1 promoters. Yeast two-hybrid and immunoprecipitation assays indicated that HSRG1 interacts with Cyclin T2, the regulatory subunit of P-TEFb, which is required for transcription elongation by RNA Pol II (RNAP II), and that amino acid residues 1-420 in Cyclin T2 are important for binding with HSRG1. Fluorescence assays suggested that the cellular localizations of those two proteins are influenced by their interaction. Further analyses with CAT assays revealed that HSRG1 inhibits the transcriptional activation by Cyclin T2 of viral promoters. Our results suggested that the inhibitory effects of HSRG1 on viral replication and proliferation are probably induced by its binding to Cyclin T2. Therefore, it is likely that HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2.

Characterization and comparison of the full 3′ and 5′ untranslated genomic regions of diverse isolates of infectious salmon anaemia virus by using a rapid and universal method

The 3′ and 5′ untranslated regions (UTRs) of the gene segments of orthomyxoviruses interact closely with the polymerase complex and are important for viral replication and transcription regulation. Despite this, the 3′ and 5′ RNA UTRs of the infectious salmon anaemia virus (ISAV) genome have only been partially characterized and little is known about the level of conservation between different virus subtypes. This report details for the first time, the adaptation of a rapid method for the simultaneous characterization of the 3′ and 5′ UTRs of each viral segment of ISAV. This was achieved through self circularization of segments using T4 RNA ligase, followed by PCR and sequencing. Dephosphorylation of 5′ ends using tobacco acid pyrophosphatase (TAP) proved to be a specific requirement for ligation of ISAV ends which was not essential for characterization of influenza virus in a similar manner. The development of universal primers facilitated the characterization of 4 genetically distinct ISAV isolates from Canada, Norway and Scotland. Comparison of the UTR regions revealed a similarity in organization and presence of conserved terminal sequences as reported for other orthomyxoviruses. Interestingly, the 3′ ends of ISAV segments including segments 1, 5 and 6, were shorter and 5′ UTRs generally longer than in their influenza counterparts.

Herpes Simplex Virus 1 ICP4 Forms Complexes with TFIID and Mediator in Virus-Infected Cells

The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. Since viral genes are transcribed by cellular RNA polymerase II (RNA pol II), ICP4 must interact with components of the pol II machinery to regulate viral gene expression. It has been shown previously that ICP4 interacts with TATA box-binding protein (TBP), TFIIB, and the TBP-associated factor 1 (TAF1) in vitro. In this study, ICP4-containing complexes were isolated from infected cells by tandem affinity purification (TAP). Forty-six proteins that copurified with ICP4 were identified by mass spectrometry. Additional copurifying proteins were identified by Western blot analysis. These included 11 components of TFIID and 4 components of the Mediator complex. The significance of the ICP4-Mediator interaction was further investigated using immunofluorescence and chromatin immunoprecipitation. Mediator was found to colocalize with ICP4 starting at early and continuing into late times of infection. In addition, Mediator was recruited to viral promoters in an ICP4-dependent manner. Taken together, the data suggest that ICP4 interacts with components of TFIID and Mediator in the context of viral infection, and this may explain the broad transactivation properties of ICP4.

Replication and transcription of human papillomavirus type 58 genome in Saccharomyces cerevisiae

BackgroundTo establish a convenient system for the study of human papillomavirus (HPV), we inserted a Saccharomyces cerevisiae selectable marker, Ura, into HPV58 genome and transformed it into yeast.ResultsHPV58 genome could replicate extrachromosomally in yeast, with transcription of its early and late genes. However, with mutation of the viral E2 gene, HPV58 genome lost its mitotic stability, and the transcription levels of E6 and E7 genes were upregulated.ConclusionsE2 protein could participate in viral genome maintenance, replication and transcription regulation. This yeast model could be used for the study of certain aspects of HPV life cycle.

Activation of the PI3K–Akt pathway by human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax increases Bcl3 expression, which is associated with enhanced growth of HTLV-1-infected T cells

Bcl3 is a member of the IkappaB family that regulates genes involved in cell proliferation and apoptosis. Recent reports indicated that Bcl3 is overexpressed in HTLV-1-infected T cells via Tax-mediated transactivation, and acts as a negative regulator of viral transcription. However, the role of Bcl3 in cellular signal transduction and the growth of HTLV-1-infected T cells have not been reported. In this study, we showed that the knockdown of Bcl3 by short hairpin RNA inhibited the growth of HTLV-1-infected T cells. Although phosphatidylinositol-3 kinase (PI3K) inhibitor reduced Bcl3 expression, inactivation of glycogen synthase kinase 3 (GSK3), an effector kinase of the PI3K/Akt signaling pathway, restored Bcl3 expression in Tax-negative but not in Tax-positive T cells. Our results indicate that the overexpression of Bcl3 in HTLV-1-infected T cells is regulated not only by transcriptional but also by post-transcriptional mechanisms, and is involved in overgrowth of HTLV-1-infected T cells.

Oligomerization of Ebola Virus VP40 Is Essential for Particle Morphogenesis and Regulation of Viral Transcription

The morphogenesis and budding of virus particles represent an important stage in the life cycle of viruses. For Ebola virus, this process is driven by its major matrix protein, VP40. Like the matrix proteins of many other nonsegmented, negative-strand RNA viruses, VP40 has been demonstrated to oligomerize and to occur in at least two distinct oligomeric states: hexamers and octamers, which are composed of antiparallel dimers. While it has been shown that VP40 oligomers are essential for the viral life cycle, their function is completely unknown. Here we have identified two amino acids essential for oligomerization of VP40, the mutation of which blocked virus-like particle production. Consistent with this observation, oligomerization-deficient VP40 also showed impaired intracellular transport to budding sites and reduced binding to cellular membranes. However, other biological functions, such as the interaction of VP40 with the nucleoprotein, NP, remained undisturbed. Furthermore, both wild-type VP40 and oligomerization-deficient VP40 were found to negatively regulate viral genome replication, a novel function of VP40, which we have recently reported. Interestingly, while wild-type VP40 was also able to negatively regulate viral genome transcription, oligomerization-deficient VP40 was no longer able to fulfill this function, indicating that regulation of viral replication and transcription by VP40 are mechanistically distinct processes. These data indicate that VP40 oligomerization not only is a prerequisite for intracellular transport of VP40 and efficient membrane binding, and as a consequence virion morphogenesis, but also plays a critical role in the regulation of viral transcription by VP40.

Both matrix proteins of Ebola virus contribute to the regulation of viral genome replication and transcription

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans and non-human primates. While the role of the EBOV major matrix protein VP40 in morphogenesis is well understood, nothing is known about its contributions to the regulation of viral genome replication and/or transcription. Similarly, while it was reported that the minor matrix protein VP24 impairs viral genome replication, it remains unclear whether it also regulates transcription, since all common experimental systems measure the combined products of replication and transcription. We have developed systems that allow the independent monitoring of viral transcription and replication, based on qRT-PCR and a replication-deficient minigenome. Using these systems we show that VP24 regulates not only viral genome replication, but also transcription. Further, we show for the first time that VP40 is also involved in regulating these processes. These functions are conserved among EBOV species and, in the case of VP40, independent of its budding or RNA-binding functions.

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The Epstein-Barr virus replication and transcription activator, Rta/BRLF1, induces cellular senescence in epithelial cells

Epstein-Barr Virus (EBV) replication and transcription activator (Rta/BRLF1) is an immediate-early transcription factor that controls the conversion of the latent viral genome into one undergoing lytic replication. By using a doxycycline-inducible expression system, the present study demonstrates that EBV Rta efficiently elicits growth arrest in the human epithelial cell line HEK293. In cells arrested by EBV Rta, the expression of p21 (CDKN1A), p27 (CDKN1B) and cyclin E were increased. In contrast, the levels of cyclin D1, CDK4 and CDK6 were sharply decreased. Activation of the host cell DNA damage response (DDR), indicated by the increasing phosphorylation of H2AX and p53 Ser15, was observed on day 3 and day 5 after EBV Rta expression, respectively. Finally, EBV Rta arrested cells exhibited strong senescence-associated β-galactosidase staining on day 10 after doxycycline induction. Together, these results indicate that, in addition to triggering viral lytic replication in epithelial cells, EBV Rta concurrently initiates a cellular senescence program that was previously undocumented. This finding, showing Rta may be centrally involved in inducing a host cell state amenable to efficient viral reproduction, in addition to its previously characterized regulation of viral transcription, provides new perspectives in understanding EBV pathogenesis.

Molecular modification of a HSV-1 protein and its associated gene transcriptional regulation

The molecular modifications of Herpes Simplex Virus Type I (HSV-1) proteins represented by acetylation and phosphorylation are essential to its biological functions. The cellular chromatin-remodeling/assembly is involved in HSV-1 associated gene transcriptional regulation in human cells harboring HSV-1 lytic or latent infections. Further investigation on these biological events would provide a better understanding of the mechanisms of HSV-1 viral gene transcriptional regulation.

HIV Evades RNA Interference Directed at TAR by an Indirect Compensatory Mechanism

HIV can rapidly evolve when placed under selective pressure, including immune surveillance or the administration of antiretroviral drugs. Typically, a variant protein allows HIV to directly evade the selective pressure. Similarly, HIV has escaped suppression by RNA interference (RNAi) directed against viral RNAs by acquiring mutations at the target region that circumvent RNAi-mediated inhibition while conserving necessary viral functions. However, when we directed RNAi against the viral TAR hairpin, which plays an indispensable role in viral transcription, resistant strains were recovered, but none carried a mutation at the target site. Instead, we isolated several strains carrying promoter mutations that indirectly compensated for the RNAi by upregulating viral transcription. Combining RNAi with the application of an antiviral drug blocked replication of such mutants. Evolutionary tuning of viral transcriptional regulation may serve as a general evasion mechanism that may be targeted to improve the efficacy of antiviral therapy.

Identification of Amino Acid Substitutions in Avian Influenza Virus (H5N1) Matrix Protein 1 by Using Nanoelectrospray MS and MS/MS

Matrix protein 1 (M1), the major structural protein of the avian influenza virus, plays a critical role in regulation of viral RNA transcription via interaction with RNA and transportation of RNP cores. Mutations in M1 have been frequently observed in the highly virulent avian influenza H5N1 virus, which might be crucial to the pathogenic function. Here we report the characterization of mutated peptides in M1 purified from highly pathogenic avian influenza virus H5N1 by nanoelectrospray MS and MS/MS analyses on a quadrupole-time-of-flight mass spectrometer (Q-TOFMS). The specificity of tandem mass spectrometry allowed the identification of six amino acid (AA) substitutions in M1, including R95K, A166V, I168T, N207S, N224S, and R230K. Two commonly observed modifications such as oxidation and deamidation were accurately assigned in the protein. Bioinformatics analysis suggested some relationship between the amino acid substitution and structural property of M1 protein. Discussions on de novo sequencing of MS/MS spectra, especially in dealing with the AA substitutions, were provided.