Extracts Of Uninfected Cells Research Articles

Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

Pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed.

Activation of the 2-5OAS/RNase L pathway in CVB1 or HAV/18f infected FRhK-4 cells does not require induction of OAS1 or OAS2 expression

The latent, constitutively expressed protein RNase L is activated in coxsackievirus and HAV strain 18f infected FRhK-4 cells. Endogenous oligoadenylate synthetase (OAS) from uninfected and virus infected cell extracts synthesizes active forms of the triphosphorylated 2-5A oligomer (the only known activator of RNase L) in vitro and endogenous 2-5A is detected in infected cell extracts. However, only the largest OAS isoform, OAS3, is readily detected throughout the time course of infection. While IFNβ treatment results in an increase in the level of all three OAS isoforms in FRhK-4 cells, IFNβ pretreatment does not affect the temporal onset or enhancement of RNase L activity nor inhibit virus replication. Our results indicate that CVB1 and HAV/18f activate the 2-5OAS/RNase L pathway in FRhK-4 cells during permissive infection through endogenous levels of OAS, but contrary to that reported for some picornaviruses, CVB1 and HAV/18f replication is insensitive to this activated antiviral pathway.

Quick, Cheap, and Easy Method for HIV-1 Human Blood Screening.

When performed under strict conditions of temperature and pH, Perchloric acid (PCA) extraction is claimed to be a reliable method of providing cytosolic composition without quantitative or qualitative changes. We applied this technique to human lymphocytes, and obtained 31P-NMR spectra of both infected and uninfected cell extracts. Our studies show that the phosphomonoester (PME) to phosphodiester (PDE) ratio in uninfected cell extracts is up to three times higher than in extracts of HIV-1 infected cells. While PME signals include mainly phosphocholine and phosphoethanolamine, the PDE was identified mainly as glycerophosphorylcholine. Changes are clearly visible within 8–12 hours after infection. We conducted our initial studies on cells infected in vitro with HIV-1. We used the cell lines U87, MAGI, SupT1 and CEM, and primary lymphocytes isolated from blood of healthy donors (obtained from Bergen Community Regional Blood Center, Paramus, NJ) infected in vitro with HIV-1 type IIIB (X4-type) or 89.6 (X4/R5-type) at an M.O.I. of 0.01. The infection of these cells was confirmed by PCR analysis. The mean PDE/PME ratio of 30 uninfected lymphocyte samples was 0.482 ± 0.094 while the mean PDE/PME ratio of 30 infected samples was 0.206±0.056. Prior to extraction all cells were isolated from dead ones using Ficoll procedure, then cell pellets containing at least 5×106 cells were placed on ice and extracted with PCA. Subsequently, the KHCO3 neutralized extract was analyzed using phosphorus NMR spectroscopy. The mechanisms that determine the content of phosphodiesters and their modulation by the presence of HIV-1 comprise another target for future investigation, but its analytical value is clearly visible. Lastly, to determine whether the observation of reduced PDE/PME ratio was applicable in the analysis of HIV-1 sero-positive patients, we analyzed, in a blinded fashion, the 31P-NMR spectra of lymphocyte extracts obtained from six HIV-1 infected individuals. HIV-1 infected cells and plasma were isolated from subjects enrolled in the AIDS clinic at the University of Durban, South Africa. These individuals constitute both HIV-1 infected asymptomatic and AIDS patients, and were previously untreated by any regimen for HIV-1 infection. This population was confined to individuals aged 18–50 and consisted of ∼50% male participants, of which 100% were Black. The mean PDE/PME ratio for these samples was 0.203 ± 0.076, and again all six samples fell below the mean PDE/PME ratio of uninfected cells minus Standard Deviation. Statistical analysis was then conducted among all of these groups: uninfected (group 1), in-vitro infected (group 2) and in-vivo infected (group 3), using the Student t-test for unpaired samples. For group 1 vs. group 2 the t = 5.34, probability = 0.000040, for group 2 vs. group 3 the t = 0.142, probability = 0.89, and for group 1 vs. group 3 the t = 4.88, probability = 0.00011. The method developed here is self-calibrating (thus it measures the ratio of two blood extract components), easy to implement, and allows for measurements to be done automatically, using methods which are much more sensitive than NMR spectroscopy- for example HPLC. This technology is also much faster and cheaper than any currently used screening technique.

Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Echinacea extracts modulate the production of multiple transcription factors in uninfected cells and rhinovirus-infected cells

Extracts of Echinacea purpurea are widely used for the prevention and treatment of common colds, coughs, bronchitis and other upper respiratory infections, many of which are caused by rhinoviruses (RVs). Recent reports have indicated that rhinoviruses can stimulate the release of various pro-inflammatory cytokines and chemokines from cultured nasal and bronchial human epithelial cells, and several transcription factors (TFs) have been implicated in this process. The effects of Echinacea treatment and rhinovirus infection on the activation of a range of transcription factors were evaluated by means of a protein/DNA array analysis. The BEAS-2B cell line was used as the model, and nuclear extracts of uninfected cells and rhinovirus-14 infected cells were examined with and without treatment with one of two chemically different Echinacea extracts. It was found that both Echinacea extracts increased the nuclear content of more than 30 transcription factors, including the 12 pro-inflammatory factors examined, such as NFkB, AP-1, AP-2 and STATs 1-6. Virus infection resulted in a more dramatic increase in these same TFs. However, when RV-infected cells were treated with either of the two Echinacea extracts, TF levels were reduced to low levels, although the pattern of the reductions was different for the two extracts. These results indicate that rhinovirus infection of epithelial cells, and treatment with Echinacea extracts, led to profound effects on numerous transcription factors, which could explain the previously observed modulation of secreted cytokines and chemokines, as well as other signaling pathways. In addition, the results could help to explain the beneficial effects of Echinacea consumption.

Vaccinia Virus Intermediate Stage Transcription Is Complemented by Ras-GTPase-activating Protein SH3 Domain-binding Protein (G3BP) and Cytoplasmic Activation/Proliferation-associated Protein (p137) Individually or as a Heterodimer

Transcription of the DNA genome of vaccinia virus occurs in the cytoplasm and is temporally programmed by early, intermediate, and late stage-specific transcription factors in conjunction with a viral multisubunit RNA polymerase. The RNA polymerase, capping enzyme, and three factors (VITF-1, VITF-2, and VITF-3) are sufficient for in vitro transcription of a DNA template containing an intermediate stage promoter. Vaccinia virus intermediate transcription factor (VITF)-1 and -3 are virus-encoded, whereas VITF-2 was partially purified from extracts of uninfected HeLa cells. Using purified and recombinant viral proteins, we showed that the HeLa cell factor was required for transcription of linear or nicked circular templates but not of super coiled DNA. HeLa cell polypeptides of approximately 110 and 66 kDa copurified with VITF-2 activity through multiple chromatographic steps. The polypeptides were separated by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry as Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and p137, recently named cytoplasmic activation/proliferation-associated protein-1. The co-purification of the two polypeptides with transcription-complementing activity was confirmed with specific antibodies, and their association with each other was demonstrated by affinity chromatography of tagged recombinant forms. Furthermore, recombinant G3BP and p137 expressed individually or together in mammalian or bacterial cells complemented the activity of the viral RNA polymerase and transcription factors. The involvement of cellular proteins in transcription of intermediate stage genes may regulate the transition between early and late phases of vaccinia virus replication.

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro.

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

A cellular protein binds vaccinia virus late promoters and activates transcription in vitro.

Available evidence indicates that the transcription of the late class of vaccinia virus genes requires the participation of several virus-encoded proteins in addition to the viral RNA polymerase. In this report we describe the identification of a protein present in extracts of uninfected HeLa cells that binds avidly to viral late promoter DNA. The protein bound specifically to several different vaccinia virus late promoters but not an early nor an intermediate promoter. DNase I footprinting localized the protein's binding site to nucleotides surrounding the transcriptional start site of the I1L promoter. Optimal promoter binding required sequences in the highly conserved TAAAT motif at the transcriptional start site as well as sequences immediately upstream; however, one variation on the motif's sequence did not affect promoter binding by the protein. Partially purified late promoter binding protein (LPBP) was capable of stimulating the transcription activity of extracts depleted of LPBP on a late promoter-driven template, establishing LPBP as a transcription activator in vitro. These results suggest that a cellular protein is responsible for targeting vaccinia virus late promoters for initiation of transcription.

The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity.

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5'-TAAAAA-3' (pac 1) and 5'-TTTTAT-3' (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.

RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 alphabetagamma for its activity.

An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be beta and gamma subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the alpha subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and betagamma subunits associate with the L(alpha) complex. Further purification of L(alpha) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of betagamma subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(alpha) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Qbeta RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff ( vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine ( P<0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.

Role of the adenovirus DNA-binding protein in in vitro adeno-associated virus DNA replication.

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.

Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro.

The nucleocapsid protein (N) and phosphoprotein (P) genes of vesicular stomatitis virus (VSV), Indiana serotype, were coexpressed in Escherichia coli BL21(DE3) by using the expression vector pET-3a. The coexpression resulted in the formation of N-P complex. The purified N-P complex was found to inhibit transcription in vitro mediated by viral ribonucleoprotein (RNP) complex in a dose-dependent manner. However, addition of uninfected mammalian cell extracts together with the N-P complex to the transcribing RNP resulted in the synthesis of full-length negative-strand genome RNA. These results indicate that the N-P complex regulated transcription and a cellular factor(s) in combination with the N-P complex may switch the RNA polymerase from transcription to replication mode.

RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.

Multiple Forms of tRNALys3in HIV-1

tRNALys3is the primer for HIV-1 reverse transcriptase (RT) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNALys3, multiple forms of tRNALys3are detected in the virus released into the cell culture media. These tRNALys3isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analysis using a tRNALys3-specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNALys3to be the major abundance form of tRNALys3in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNALys3species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3′ end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNALys3with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNALys3. These radioactive tRNALys3species retained their relative mobilities when rechromatograped on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNALys3. These results indicate that the major tRNALys3species present in the virus is also the major tRNALys3isoacceptor used as the primer for reverse transcription.

In vitro replication of adeno-associated virus DNA: enhancement by extracts from adenovirus-infected HeLa cells.

Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstructural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine replication of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is 50-fold greater in extracts from Ad-infected cells than in extracts from uninfected cells. In addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated strands, whereas the majority of labeled full-length AAV DNA molecules synthesized in the uninfected extract have only one newly replicated strand. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the case of the Ad-infected cell extract is much more likely to result in the synthesis of a full-length AAV DNA molecule. Most of the newly replicated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provides a helper function related to elongation during replication by a single-strand displacement mechanism. In the assay using the uninfected HeLa cell extract, replication frequently stalls before reaching the end of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template. In support of this conjecture, replication in the uninfected cell extract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA. In addition, when shorter substrate molecules are used as the template in the uninfected HeLa cell assay, a greater proportion of the labeled full-length substrate molecules contain two newly replicated strands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad-infected 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.

Transcriptional Activation of RNA Polymerase III-Dependent Genes by the Human T-Cell Leukemia Virus Type 1 Tax Protein

The human T-cell leukemia virus-encoded tax protein is a potent activator of many viral and cellular genes transcribed by RNA polymerase II. We find that both chromatin and cell extracts derived from human T-cell leukemia virus type 1-infected human T lymphocytes support higher levels of 5S rRNA and tRNA gene transcription than chromatin or extracts from uninfected T lymphocytes. The viral protein Tax was likely responsible for this higher level of class II gene transcription, as purified Tax was found to stimulate both genes when added to the uninfected cell extract or in reconstituted systems. Both limiting-component transcription assays and DNA binding assays identified the class III gene transcription factor TFIIIB as the principle target of Tax activity. Surprisingly, we find that Tax increases the effective concentration of active TFIIIB molecules. These data suggest that Tax stimulates RNA polymerase III-dependent gene expression by accelerating the rate and/or extent of transcription initiation complex assembly.