Host Cell RNA Polymerase Research Articles

Mitotic deacetylase complex (MiDAC) recognizes the HIV-1 core promoter to control activated viral gene expression.

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.

ICP22/IE63 Mediated Transcriptional Regulation and Immune Evasion: Two Important Survival Strategies for Alphaherpesviruses.

In the process of infecting the host, alphaherpesviruses have derived a series of adaptation and survival strategies, such as latent infection, autophagy and immune evasion, to survive in the host environment. Infected cell protein 22 (ICP22) or its homologue immediate early protein 63 (IE63) is a posttranslationally modified multifunctional viral regulatory protein encoded by all alphaherpesviruses. In addition to playing an important role in the efficient use of host cell RNA polymerase II, it also plays an important role in the defense process of the virus overcoming the host immune system. These two effects of ICP22/IE63 are important survival strategies for alphaherpesviruses. In this review, we summarize the complex mechanism by which the ICP22 protein regulates the transcription of alphaherpesviruses and their host genes and the mechanism by which ICP22/IE63 participates in immune escape. Reviewing these mechanisms will also help us understand the pathogenesis of alphaherpesvirus infections and provide new strategies to combat these viral infections.

COVID-19 vaccines: Where do we stand?

Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first reported in the city Wuhan, China in December 2019. The high rates of infection led to quick spread of the virus around the world and on March 11th, 2020, the World Health Organization (WHO) announced the pandemic of the Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2. The pharmaceutical companies and institutions have been working towards developing a safe and effective vaccine in order to control the pandemic. The biology of SARS-CoV-2 is briefly discussed describing the transcription of the virus and the receptor recognition. The spike protein of SARS-CoV-2 is important in the attachment of the host cell and RNA-dependent RNA polymerase (RdRp) is involved in the replication of the virus making them good candidates for drug and vaccine targets. To date many different strategies have been employed in the development of vaccines and a number of them are in the phase III of clinical trials with promising results. In this mini-review, we assessed the literature throughly and described the latest developments in SARS-CoV-2 vaccines for humans. The main benefits and drawbacks of each platform is evaluated and the possible changes in the vaccine effectivity due to naturally occuring SARS-CoV-2 mutations have been described.

Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates

Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cancer development. HBV encodes the hepatitis B virus X (HBx) protein that promotes transcription of the viral episomal DNA genome by the host cell RNA polymerase II. Here we provide evidence that HBx accomplishes this task by a conserved and unusual mechanism. Thus, HBx strongly stimulates expression of transiently transfected reporter constructs, regardless of the enhancer and promoter sequences. This activity invariably requires HBx binding to the cellular UV-damaged DDB1 E3 ubiquitin ligase, suggesting a common mechanism. Unexpectedly, none of the reporters tested is stimulated by HBx when integrated into the chromosome, despite remaining responsive to their cognate activators. Likewise, HBx promotes gene expression from the natural HBV episomal template but not from a chromosomally integrated HBV construct. The same was observed with the HBx protein of woodchuck HBV. HBx does not affect nuclear plasmid copy number and functions independently of CpG dinucleotide methylation. We propose that HBx supports HBV gene expression by a conserved mechanism that acts specifically on episomal DNA templates independently of the nature of the cis-regulatory sequences. Because of its uncommon property and key role in viral transcription, HBx represents an attractive target for new antiviral therapies.

Crystal structure of HIV-1 Tat complexed with human P-TEFb.

Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat•P-TEFb complex containing HIV-1 Tat, human Cdk9, and human Cyclin T1. Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the Cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat•P-TEFb complex and block HIV replication.

Identification of Sequences in Herpes Simplex Virus Type 1 ICP22 That Influence RNA Polymerase II Modification and Viral Late Gene Expression

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

DNA-replication complex from cells infected with herpes virus.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain length of 25 nucleotides, to which the newly synthesized DNA is covalently attached. The chemical nature of the RNA primer was proven by degradation experiments with endoribonuclease V. In the absence of any initiator in the reaction mixture the HSV-induced DNA polymerase is inactive. Kinetic experiments revealed that DNA synthesis in assays containing poly(dT), dNTPs and NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA replication complex was further fractionated into HSV-induced DNA polymerase and the three cellular RNA polymerases. In the reconstructed system DNA synthesis, catalyzed by virus-induced DNA polymerase starts in a reaction assay, containing single-stranded DNA template, only in the presence of RNA polymerase II. The conclusion that the subnucleoplasmic complex, isolated from HSV-infected cells, is of biological significance for HSV DNA synthesis stems (a) from the the close correlations of the known biochemical events occurring during the initiation of HSV DNA synthesis in intact cells and in the nucleoplasmic system and (b) from control experiments with a subnucleoplasmic complex, isolated from uninfected cells, which is devoid of any potency to initiate DNA synthesis.

Efficient transcription of the EBER2 gene depends on the structural integrity of the RNA.

A 3'-truncated EBER2 RNA gene, although containing all previously identified promoter elements, revealed drastically reduced transcription rates in vitro and in vivo when fused to a heterologous terminator sequence. Inactivations were also observed with double point mutations affecting 5'- or 3'-end sequences of the EBER2 gene. However, wild-type activity of these mutants could be restored by compensatory mutations of the opposite strand of the EBER2 RNA sequence. A similar rescue was achieved with the 3'-truncated EBER2 gene, if the heterologous terminator was adapted for complementarity to the initiator element of the construct. Yet, double-strandedness alone of the RNA ends was not sufficient for high transcriptional activity of these gene constructs. Rather, the use of a nonrefoldable spacer, separating the 5'- and 3'-stem-loop structures, demonstrated that spatial proximity of the ends of EBER2 RNA was required. Furthermore, decay kinetics of wild-type and mutant RNA synthesized in vitro indicated that the effects observed could not be explained by altered transcript stability. Finally, single-round transcription confirmed that the reduced expression of mutant genes was not caused by decreased primary initiation reactions. In addition, differential sarcosyl concentrations demonstrated that the rate of reinitiation clearly was affected with the mutant EBER2 genes. Together, these results indicate that the secondary structure of this viral RNA represents a major determinant for efficient transcription of the EBER2 gene by host cell RNA polymerase III.

Lethal mutagenesis of HIV by mutagenic ribonucleoside analogs.

The extraordinarily high mutation rate of HIV suggests that the viral population exists near the threshold for viral viability. An increase in mutation could mediate a lethal increase in the already high proportion of defective viruses. We initially tested this hypothesis by culturing HIV-infected cells in mutagenic deoxyribonucleoside analogs. In the presence of 5-hydroxydeoxycytidine, there was a loss of viral replication after sequential passages of HIV in human CEM cells (Loeb LA, et al.: Proc Natl Acad Sci USA 1999;96:14921497). We now propose to use mutagenic ribonucleosides to induce lethal mutagenesis. The incorporation of mutagenic analogs into the HIV genome by the host cell RNA polymerase, rather than by the viral reverse transcriptase, augurs well for the avoidance of resistant viruses.

Poliovirus-encoded protease 2APro cleaves the TATA-binding protein but does not inhibit host cell RNA polymerase II transcription in vitro.

Transient expression of the poliovirus-encoded protease 2APro in eukaryotic cells results in inhibition of both cellular transcription and translation. The inhibition of transcription observed in cells expressing 2APro could be due to a primary effect or secondary effect caused by inhibition of translation. Because transcriptional activity of the TATA-binding protein (TBP) is drastically reduced in poliovirus-infected cells, we determined if 2APro is able to cleave TBP in vitro. We demonstrate here that 2APro directly cleaves the single tyrosine-glycine bond at position 34 of TBP. This cleavage is also seen in poliovirus-infected HeLa cells. Surprisingly, despite TBP cleavage 2APro was unable to inhibit RNA polymerase II transcription in vitro. Under similar conditions, however, 2APro inhibited translation of a capped cellular mRNA in vitro. Thus, cleavage of TBP at position 34 does not alter its transcriptional activity in vitro. These results suggest that inhibition of host cell RNA polymerase II transcription seen in cells transiently transfected with 2APro is due to host cell translational shutoff.

Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro.

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. Previous studies from our laboratory showed that activated transcription from a cyclic AMP-responsive element (CRE)-containing promoter was severely inhibited in extracts prepared from poliovirus-infected HeLa cells compared to those from mock-infected cells. Here we demonstrate that the CRE-binding protein, CREB, is specifically cleaved by the poliovirus-encoded protease 3Cpro both in vitro and in virus-infected cells. The proteolytic cleavage of CREB leads to a significant loss of its DNA binding as well as transcriptional activity. Additionally, we demonstrate that the phosphorylated, transcriptionally active form of CREB is cleaved by the viral protease in vitro. The results presented here suggest that a direct cleavage of CREB by the viral protease 3Cpro leads to inhibition of CREB-activated transcription in poliovirus-infected HeLa cells.

Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro.

Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.

RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection.

During lytic infection, herpes simplex virus subverts the host cell RNA polymerase II transcription machinery to efficiently express its own genome while repressing the expression of most cellular genes. The mechanism by which RNA polymerase II is directed to the viral delayed-early and late genes is still unresolved. We report here that RNA polymerase II is preferentially localized to viral replication compartments early after infection with herpes simplex virus type 1. Concurrent with recruitment of RNA polymerase II into viral compartments is a rapid and aberrant phosphorylation of the large subunit carboxy-terminal domain (CTD). Aberrant phosphorylation of the CTD requires early viral gene expression but is not dependent on viral DNA replication or on the formation of viral replication compartments. Localization of RNA polymerase II and modifications to the CTD may be instrumental in favoring transcription of viral genes and repressing specific transcription of cellular genes.

Identification of the cleavage site and determinants required for poliovirus 3CPro-catalyzed cleavage of human TATA-binding transcription factor TBP

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. We have shown previously that the human TATA-binding protein (TBP), a general transcription factor required for transcription of all RNA polymerase II genes, is directly cleaved both in vitro and in vivo by the virus-coded protease 3CPro. 3CPro specifically cleaves glutamine-glycine bonds in the viral polyprotein. Cellular transcription factor TBP contains three glutamine-glycine sites, at amino acids 12, 18, and 108. By using site-directed mutagenesis, we determined that the glutamine-glycine bond at amino acid 18, but not that at amino acid 12 or 108, is cleaved by the viral protease. Both the glutamine and the glycine appear to be important for the cleavage. Further mutations around the glutamine-glycine site at position 18 suggest that determinants other than the glutamine-glycine bond in TBP are also required for 3CPro-induced cleavage. An alanine at position P4 and a proline at position P2, proximal to the scissile glutamine-glycine pair, appear to be important for 3CPro-mediated cleavage of TBP. Our results suggest that the cleavage specificity of 3CPro for a cellular transcription factor is very similar to its mode of cleavage of viral polyproteins.

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

Replication strategy and pathogenesis of HDV

Hepatitis delta virus (HDV) is a defective human pathogen which is dependent on hepatitis B virus (HBV) for replication and expression. The full extent of the helper function is still unknown, but HBV provides a hepatitis B surface antigen envelope that surrounds teh HDV-specific internal components of the HDV virion. These components are a circular single-stranded RNA genome of approximately 1.7kb and hepatitis delta antigen (HDAg). The genome has five potential genes, but a protein product (HDAg) has only been identified from one. HDAg is found in the liver and serum of infected individuals as two related polypeptides of 24- and 27-kDa. The genome also shares a number of similarities with the viroids, highly pathogenic agents of plants including (1) the ability for RNA self-cleavage in vitro ; (2) genome replication by the rolling circle mechanism; (3) nuclear replication by host cell RNA polymerase II. A number of studies have shown that HDV RNA replication is dependent on HDAg; however recent evidence suggests that the role of HDAg is to transport HDV RNA to the nucleus but it does not form part of the transcriptional complex per se . Virus replication is generally associated with cell death, both in vivo and in vitro , that may be a result of the expression of HDAg p24 which appears to inhibit host cell RNA synthesis.

Creation of a T7 autogene: Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter

The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer, T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.

Transcription of methylated viral DNA by eukaryotic RNA polymerase II

The genome of the large icosahedral DNA virus, frog virus 3 (FV3), is heavily methylated at the cytosine residues of dCdG dinucleotide pairs, with more than 22% of the total cytosine residues in the form of 5-methylcytosine (5mC). This methylation is carried out postreplicatively in the cytoplasm of infected cells by a virus-encoded DNA methyltransferase. DNA methyltransferase activity was shown to copurify with a 26 kD virus-induced, DNA-binding protein that had an altered mobility in extracts from cells infected with a DNA-methyl-transferase deficient mutant of FV3. Immediately after infection, the highly methylated parental DNA is transcribed in the nucleus by the host cell RNA polymerase II. As FV3 induces the synthesis of a protein that can override the inhibitory effect of methylation on the transcription of exogenous promoters methylation in vitro, we suggest that this protein is a factor evolved by this virus to allow transcription from methylated promoters by eukaryotic RNA polymerase II.

Poliovirus-induced modification of host cell RNA polymerase IIO is prevented by cycloheximide and zinc.

Infection of HeLa cells with poliovirus results in a decrease in the level of RNA polymerase IIO, the transcriptionally active form of the enzyme, and a shutdown of host transcription (Rangel, L. M., Fernández-Tomas, C., Dahmus, M. E., and Gariglio, P. (1987) J. Virol. 61, 1002-1006). The effect of cycloheximide on poliovirus-induced modification of host RNA polymerase IIO was investigated. The inhibition of protein synthesis, at sequential stages during viral replication, prevents the modification of both total and chromatin-bound RNA polymerase IIO. Furthermore, the inclusion of zinc at a concentration that inhibits the proteolytic post-translational processing of viral polyprotein also prevents the modification of RNA polymerase IIO. These results suggest that host cell enzyme modification depends on the synthesis and processing of protein(s) encoded by the viral genome.