Pol mRNA Research Articles

Proteomic analysis of Streptomyces coelicolor in response to Ciprofloxacin challenge

Multi-drug tolerance is an important phenotypic property that complicates treatment of infectious diseases and reshapes drug discovery. Hence a systematic study of the origins and mechanisms of resistance shown by microorganisms is imperative. Since soil-dwelling bacteria are constantly challenged with a myriad of antibiotics, they are potential reservoirs of resistance determinants that can be mobilized into pathogens over a period of time. Elucidating the resistance mechanisms in such bacteria could help future antibiotic discoveries. This research is a preliminary study conducted to determine the effects of ciprofloxacin (CIP) on the intrinsically resistant Gram-positive soil bacterium Streptomyces coelicolor. The effect was investigated by performing 2-DE on total protein extracts of cells exposed to sub-lethal concentrations of ciprofloxacin as compared to the controls. Protein identification by MALDI-TOF/TOF revealed 24 unique differentially expressed proteins, which were statistically significant. The down-regulation of proteins involved in carbohydrate metabolism indicated a shift in the cell physiology towards a state of metabolic shutdown. Furthermore, the observed decline in protein levels involved in transcription and translation machinery, along with depletion of enzymes involved in amino acid biosynthesis and protein folding could be a cellular response to DNA damage caused by CIP, thereby minimizing the effect of defective and energetically wasteful metabolic processes. This could be crucial for the initial survival of the cells before gene level changes could come into play to ensure survival under prolonged adverse conditions. These results are a first attempt towards profiling the proteome of S. coelicolor in response to antibiotic stress. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. Soil-dwelling bacteria could serve as a reservoir of resistance determinants for clinically important bacteria. In this work, we investigated, for the first time, the differential proteomic profile of S. coelicolor cells in response to sub-inhibitory concentrations of Ciprofloxacin using 2-DE. Results indicate a shift in the cell physiology towards a state of metabolic shutdown, possibly to counter the DNA damage by ciprofloxacin. Further, up-regulation of GAPDH, RNA pol mRNA and Translation IF2 protein indicates a reprogramming of the cell for long-term survival. This study could serve as a basis for further investigations to elucidate the general mechanism by which soil bacteria exhibit resistance to fluroquinolones. This may help in developing new drug protocols and inventing novel drugs to counter resistance to this class of antibiotics in pathogenic bacteria.

Abstract A62: Human endogenous retrovirus activation in prostate cancer: Association with disease progression

Abstract Introduction: In recent years scientists have observed that several tumor types, including prostate cancer, show increased expression of human endogenous retrovirus (HERV) when compared to normal tissue. The HERVs originated from germ cell infections by exogenous retroviruses during the course of evolution and became incorporated into the human genome. These elements are widely dispersed throughout the genome and are estimated to comprise of greater than >8% of genomic material. The HERV-K family, evolutionarily the youngest HERV, is the only HERV family with complete open reading frames for all viral genes, and thus are the most likely to be biologically active and potentially pathogenic. The HERV-K provirus encodes for the gag, pol and env genes flanked by two long terminal repeat regions. Both HERV-K type I and HERV-K type II proviruses exist: Type II proviruses are complete (and a such are chronological “older” with regard to their insertion into the genome), while Type I proviruses contain a 292-bp deletion at the boundary of the pol and env genes. Hypothesis: We hypothesize that HERV-K expression is activated with advancing prostate cancer progression and that as a result of this, HERV-K leads to a sustained inflammatory response and subsequent promotion of tumor progression. We believe that HERV-K is suitable target of therapeutic intervention. Experimental Procedures: RNA sequences for type II HERV-K108 (AF074086) were blasted against type I HERV-K102 (AF164610). Sequences were 99% homologous between nucleotides 1-6492 (K108) & 1-1691 (K102) (5′LTR-gag-pro-pol region), the type 1 del in K102 occurred between nucleotides 6493-6793 of K108, subsequent nucleotides 6794-9472 (K108) & 6501-9178 (K102) were 99% homologous (env-3′LTR region). For the detection of HERV-K gag, pol and env mRNA proteins were designed in Primer3, and verified for specificity in Primer-BLAST. Levels of HERV-K gag, pol and env type I or II mRNA were quantified in RWPE1 normal immortalized prostate cells, and CRW22 androgen dependent prostate cancer cell lines, and 22Rv1, PC-3 and DU145 androgen independent prostate cancer cell lines (22Rv1 AI variant of CRW22, PC-3 and DU145 derived from prostate cancer metastasis). HERV-K gag and env protein expression was detected in cell lines by western blotting and in prostate cancer tissue microarrays of hyperplasia and adenocarcinoma using anti-HERV-K gag monoclonal antibody (MAb) and anti-HERV-K env MAb. Effects of HERV-K env inhibition in prostate cancer cells were examined by inhibiting HERV-K env using a HERV-K env targeted MAb, and assessing its effect on cell proliferation and cell invasion using the xCelligence Real-Time Cell Analyser. Results: We found that HERV-K is activated in CRW22, 22Rv1, PC3 and DU145 prostate cancer cell lines, but not in RWPE1 normal prostate cells. Additionally, we observed the HERV-K levels were highest in the bone metastasis derived PC3 and brain metastasis derived DU145 cell lines. We also found that HERV-K was differentially expressed between prostate hyperplasia and adenocarcinoma. Inhibition of HERV-K env resulted in inhibition of PC3 and DU145 cell proliferation and cell invasion. Conclusions: HERV-K is activated in prostate cancer and represents a potential therapeutic target for metastatic prostate cancer. Citation Format: Ronan Downey, Amy Burke, Francis J. Giles, Frank Sullivan, Feng Wang-Johanning, Blanaid Mee, Eoin Gaffney, Sharon Anneve Glynn. Human endogenous retrovirus activation in prostate cancer: Association with disease progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A62.

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

Dichotomy of All-Trans Retinoic Acid Inducing Signals for Adult T-Cell Leukemia.

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax or pol mRNA) using the real time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT was inhibitory of proviral DNA but not NF-kB transcriptional activity and sIL-2R on HTLV-1, however ATRA was inhibitory of NF-kB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease of sIL-2R induced by ATRA may be caused by the actions of a NF-kB inhibitor acting on the NF-kB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kB inhibitor and as a RT inhibitor.

Dichotomy of all-trans retinoic acid inducing signals for adult T-cell leukemia

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in human T-cell leukemia virus type 1 (HTLV-1)-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-kappaB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax, or pol mRNA) using the real-time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT inhibited proviral DNA but not NF-kappaB transcriptional activity, and sIL-2R on HTLV-1; however, ATRA inhibited of NF-kappaB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease in sIL-2R induced by ATRA may be caused by the actions of a NF-kappaB inhibitor acting on the NF-kappaB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kappaB inhibitor and as an RT inhibitor.

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3'-truncated pgRNA lacking 3' direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.

Solution structure of the HIV-1 frameshift inducing stem-loop RNA.

The translation of reverse transcriptase and other essential viral proteins from the HIV-1 Pol mRNA requires a programmed -1 ribosomal frameshift. This frameshift is induced by two highly conserved elements within the HIV-1 mRNA: a slippery sequence comprised of a UUUUUUA heptamer, and a downstream stem-loop structure. We have determined the structure of the HIV-1 frameshift inducing RNA stem-loop, using multidimensional heteronuclear nuclear magnetic resonance (NMR) methods. The 22 nucleotide RNA solution structure [root mean squared deviation (r.m.s.d.) = 1.2 A] was determined from 475 nuclear Overhauser effect (NOE)-derived distance restrains, 20 residual dipolar couplings and direct detection of hydrogen bonds via scalar couplings. We find that the frameshift inducing stem-loop is an A-form helix capped by a structured ACAA tetraloop. The ACAA tetraloop is stabilized by an equilateral 5' and 3' stacking pattern, a sheared A-A pair and a cross-strand hydrogen bond. Unexpectedly, the ACAA tetraloop structure is nearly identical to a known tetraloop fold, previously identified in the RNase III recognition site from Saccharomyces cerevisiae.

Regulation of bovine leukemia virus tax and pol mRNA levels by interleukin-2 and -10.

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.

The Epstein-Barr virus (EBV) SM protein enhances pre-mRNA processing of the EBV DNA polymerase transcript.

The Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA, which contains a noncanonical polyadenylation signal, UAUAAA, is cleaved and polyadenylated inefficiently (S. C. S. Key and J. S. Pagano, Virology 234:147-159, 1997). We postulated that the EBV early proteins SM and M, which appear to act posttranscriptionally and are homologs of herpes simplex virus (HSV) ICP27, might compensate for the inefficient processing of pol pre-mRNA. Here we show that the SM and M proteins interact with each other in vitro. In addition, glutathione S-transferase-SM/M fusion proteins precipitate the heterogeneous ribonucleoprotein (hnRNP) C1 splicing protein. Further, the SM protein is coimmunoprecipitated from SM-expressing cell extracts with an antibody to the hnRNP A1/A2 proteins, which are splicing and nuclear shuttling proteins. Finally, the amount of processed EBV DNA polymerase mRNA was increased three- to fourfold in a HeLa cell line expressing SM; this increase was not due to enhanced transcription. Thus, inefficient processing of EBV pol RNA by cellular cleavage and polyadenylation factors appears to be compensated for and may be regulated by the early EBV protein, SM, perhaps via RNA 3'-end formation.

A Noncanonical Poly(A) Signal, UAUAAA, and Flanking Elements in Epstein–Barr Virus DNA Polymerase mRNA Function in Cleavage and Polyadenylation Assays

Two forms of the Epstein–Barr virus DNA polymerase (pol) mRNA (3.7 and 5.1 kb) have been detected, neither of which contains a canonical poly(A) signal. The 5.1-kb pol mRNA, which contains a rare poly(A) signal, UAUAAA, studied only in transcripts of Hepadnaviridae and a plant pararetrovirus, was analyzed in cleavage and polyadenylation assays. Incubation of the pol transcript in cell extracts produced relatively low efficiency of cleavage (12 to 14%), which was improved by conversion of the poly(A) signal to AAUAAA. Deletion of the UAUAAA signal abolished cleavage and polyadenylation. An auxiliary element, UUUGUA, 3–8 nt upstream of the poly(A) signal and two downstream core elements, a GU-rich sequence 36–46 nt, and an AUUUGUGU sequence 47–53 nt downstream of the signal (8–19 nt and 20–28 nt downstream of cleavage site) facilitated processing of pol mRNA. Replacement of sequences near the cleavage/poly(A) site affected cleavage accuracy. Binding of the 64-kDa cleavage stimulatory factor to the U-rich as well as the GU-rich elements correlated with cleavage efficiency. Thus the UAUAAA hexanucleotide plus the other cis-acting elements are clearly functional in the native pol mRNA, but are relatively inefficient. Implications of the use of an anomalous poly(A) signal and its elements are discussed.

A rapid and efficient system for screening HIV-1 Pol mRNA-specific ribozymes.

Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs. However, the reports that exist on their activity against different targets have described mixed success. As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzPro directed against the protease (Pro) coding region and RzRT directed against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli. Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro. RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzPro and RzRT target sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT. The RT activity in cells expressing RzRT was shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any that could enhance ribozyme activity in vivo.

Characterization of the spliced pol transcript of feline foamy virus: the splice acceptor site of the pol transcript is located in gag of foamy viruses.

Foamy viruses, or spumaviruses, are distinct members of the Retroviridae. Here we have characterized the long terminal repeat of the feline, or cat, foamy virus by determining the locations of the transcriptional start site and the poly(A) addition site. The splice donor and splice acceptor sites of the subgenomic mRNA responsible for Pro-Pol protein expression were identified by nucleotide sequencing of the corresponding cDNAs. The leader exon of the feline foamy virus is 57 nucleotides long. The splice acceptor of the subgenomic pol mRNA was found to be located in gag. The location of the splice acceptor of the human foamy virus pol mRNA was confirmed to map in gag. The pol splice acceptor site in gag of the cat foamy virus is located further downstream than that of human foamy virus.

Expression of Human Foamy Virus Reverse Transcriptase Involves a Spliced pol mRNA

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5′ splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 orders of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.

Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerases in heterologous systems. We have expressed human hepatitis B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. Analysis of immunoaffinity-purified pol indicated that (i) a portion of pol had initiated minus-strand DNA synthesis within infected insect cells; (ii) the pol mRNA appeared to be the template for reverse transcription; (iii) the products were small (100 to 500 nucleotides); (iv) only minus-strand DNA was synthesized; (v) the products were covalently bound to protein; and (vi) the 5' end of the minus-strand DNA mapped to DR1 by primer extension. The purified pol was also active in an in vitro polymerase assay. Analyses suggested that a different fraction of pol was active in the in vitro assays. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of the pol polypeptide in a reaction that appeared to represent in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of 32P-labeled phosphotyrosine on pol following in vitro reactions with 32P-labeled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will facilitate functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication.

Unusual regulation of expression of the herpes simplex virus DNA polymerase gene

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.

Regulation of the Epstein-Barr virus DNA polymerase gene.

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.

Regulation of HTLV-II gene expression by rex involves positive and negative cis-acting elements in the 5′ long terminal repeat

Regulation of human T-cell leukemia virus type II (HTLV-II) gene expression by the trans-acting viral protein, Rex, is mediated through specific cis-acting sequences in the HTLV-II long terminal repeat (LTR). Augmentation of 5′ LTR-linked gene expression by Rex requires two distinct cis-acting elements: one termed the “Rex-responsive element” (RxRE), which allows Rex to overcome the inhibitory effect of a second, termed the “ cis-acting repressive sequences” (CRS). The HTLV-II RxRE is located between nt +91 and +317 relative to the cap site in the R/U5 region of the 5′ LTR, and the HTLV-II CRS is contained within the RxRE from nt +208 to +317, which is downstream of the splice donor site, in the R/U5 region of the 5′ LTR. Deletion of the CRS results in significantly increased cytoplasmic levels of LTR-linked mRNA independent of the presence of Rex. Our results show that Rex acts post-transcriptionally and induces a shift from nucleus to cytoplasm of gag/ pol mRNA, the only HTLV-II mRNA that contains both the RxRE and the CRS in the 5′ LTR-derived leader sequence.

Differential modulation of host cell and hiv gene expression by combinations of avarol and AZT in vitro

Human immunodeficiency virus type 1 (HIV-1) gene expression is modulated by some virus-encoded proteins, possibly acting at multiple levels of control, which are also known to be involved in the regulation of gene expression in uninfected cells (transcriptional, post-transcriptional, nucleocytoplasmic transport, and translational control). Two anti-HIV-1 drugs, Avarol and 3'-azido-3'-deoxythymidine, which inhibit viral replication by differential mechanisms, were used to study the role of cytoplasmic factors in independent regulation of host cell and viral gene expression. Both drugs were found to inhibit viral replication and synthesis of virus-encoded protein in a synergistic manner, while at cytostatic concentrations, both compounds act antagonistically. ATP-induced transport of viral messengers from isolated nuclei is enhanced by total cytosolic protein from HIV-1-infected cells; a strong increase of the nucleocytoplasmic transport of pol mRNA was measured and, to a lesser extent the transport of certain cellular mRNA (e.g. interleukin-2) was augmented, while the transport of other cellular mRNA (actin) was not affected at all.

Expression of enzymatically active adenovirus DNA POLYMERASE FROM CLONED DNA requires sequences upstream of the main open reading frame

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the preterminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. Plasmids containing the putative exons that encode Ad Pol mRNA were constructed to determine if enzymatically active Ad Pol protein could be synthesized. An Ad Pol of 140 kDa was detected by immunoprecipitation with specific antibody and its enzymatic activity was confirmed by complementation of Ad DNA replication in vitro. In addition to an Ad2 DNA fragment from 24.7 to 9.2 map units which contains an open reading frame for a protein of 120 kDa, the HindIII-J fragment that encodes the axon at genome coordinate 39 can be shown to be essential for production of full-length (140 kDa), enzymatically active Ad Pol.

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.