Early Viral Protein Synthesis Research Articles

Inactivation Kinetics and Replication Cycle Inhibition of Adenovirus by Monochloramine

Monochloramine is commonly used as a secondary disinfectant to maintain a residual in drinking water distribution systems in the United States. The mechanism by which waterborne viruses become inactivated by monochloramine remains widely unknown. A more fundamental understanding of how viruses become inactivated is necessary for better detection and control of viruses in drinking water. Human adenovirus (HAdV) is known to be the waterborne virus most resistant to monochloramine disinfection, and this study presents inactivation kinetics over a range of environmental conditions. Several steps in the HAdV replication cycle were investigated to determine which steps become inhibited by monochloramine disinfection. Interestingly, monochloramine-inactivated HAdV could bind to host cells, but genome replication and early and late mRNA transcription were inhibited. We conclude that monochloramine exposure inhibited a replication cycle event after binding but prior to early viral protein synthesis.

Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

BackgroundTo understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.ResultsTo determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.ConclusionsThe F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.

Early viral protein synthesis is necessary for NF-κB activation in modified vaccinia Ankara (MVA)-infected 293 T fibroblast cells

Modified vaccinia Ankara (MVA) is an attenuated vaccinia virus, and is a promising vaccine vector for variola and monkeypox viruses, as well as for other pathogens. The MVA determinants important for vaccine efficacy and immunogenicity are poorly defined. MVA infection of fibroblast cells activates NF-kappaB, a characteristic not ascribed to wild-type vaccinia viruses. Thus, NF-kappaB activation, and the subsequent upregulation of host immune molecules, could be one of the determinants for MVA's immunogenicity. We report that ERK2 phosphorylation, an event preceding and required for NF-kappaB activation, occurred rapidly after virus infection. ERK2 and NF-kappaB remained inert when virus endocytosis was prevented, suggesting that virus-host cell interactions were insufficient for activating NF-kappaB. Inhibition of viral protein synthesis decreased NF-kappaB activation, and elimination of intermediate and late gene expression did not alter MVA-induced NF-kappaB activation. Thus, early gene expression activates NF-kappaB.

The IR4 auxiliary regulatory protein expands the in vitro host range of equine herpesvirus 1 and is essential for pathogenesis in the murine model

IR4, an early regulatory protein of equine herpesvirus 1 (EHV-1), is not a DNA-binding protein, but interacts with the sole immediate-early protein (IEP) to increase both IEP site-specific DNA-binding and IEP-mediated trans-activation of EHV-1 promoters. To investigate the biological properties of IR4 and ascertain whether this regulatory protein is essential for virus growth, bacterial artificial chromosome methods were employed to generate an IR4-null EHV-1. The IR4 gene was dispensable for EHV-1 growth in non-immortalized equine NBL-6 cells, but virus replication was delayed and was reduced by greater than 10-fold. In addition, replication of the IR4 mutant was abrogated in all other cell types tested, including equine ETCC tumor cells and cells of mouse, rabbit, monkey, and human origin. Further, in contrast to the highly pathogenic parent virus, the IR4 deletion mutant failed to cause disease in the CBA mouse as judged by assessing body weight and clinical signs and was unable to replicate in the murine lung. To define the nature of the block in the replication of the IR4-null virus, molecular analyses were carried out in RK-13 rabbits' cells infected with the IR4-deleted virus and revealed that: 1) the synthesis of the sole IEP was not inhibited; 2) the synthesis of early viral proteins examined was either not affected or was delayed to late times; 3) viral DNA replication was inhibited by more than 99.9%; and 4) synthesis of essential late proteins such as glycoprotein D and glycoprotein K was prevented. These findings indicate that the IR4 protein is required for EHV-1 DNA replication in non-permissive cells, and, like its homologues in other alphaherpesviruses, contributes a function required for virus replication in a variety of cell types.

Antiviral Activity of Lauryl Gallate against Animal Viruses

Background Antiviral compounds are needed in the control of many animal and human diseases. Methods We analysed the effect of the antitumoural drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (herpes simplex and vaccinia) and RNA (influenza, porcine transmissible gastroenteritis and Sindbis) viruses, paying attention to its effect on the viability of the corresponding host cells. Results Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1–10 μM), reducing the titres 3–>5 log units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5–8 h post-infection. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug; however, the early viral protein synthesis and the virus-mediated increase of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells. Furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. Conclusions Lauryl gallate is a powerful antiviral agent against several pathogens of clinical and veterinary importance. The overall results indicate that a cellular factor or function might be the target of the antiviral action of alkyl gallates.

Open reading frame 193R of Chilo iridescent virus encodes a functional inhibitor of apoptosis (IAP)

Programmed cell death or apoptosis is a major defense mechanism in insects in response to viral infections. The genome of Chilo iridescent virus (CIV) has three ORFs with homology to baculovirus inhibitor of apoptosis (iap) genes. The proteins encoded by the 157L, 193R, and 332L ORFs contain 152, 208 and 234 amino acids, respectively. While all three proteins contain C-terminal RING domains, only the protein encoded by ORF 193R contains a baculoviral iap repeat (BIR) domain, indicative of a putative IAP protein. The 193R protein has 28 and 27% similarity in amino acid sequence to the Orgyia pseudotsugata MNPV and Cydia pomonella granulovirus IAP-3 proteins, respectively. ORF 193R from CIV is the only gene known to exist among the Iridoviridae that encodes a BIR domain. 193R is transcribed early during CIV infection, and its transcription is not dependent on the synthesis of early viral proteins. When this putative CIV IAP was transiently expressed in SPC-BM-36 and Sf21 cells under the control of an immediate early baculovirus promoter it significantly reduced apoptosis induced by actinomycin-D. Silencing of the CIV iap gene (193R) in CIV infected SPC-BM-36 cells with 193R-specific dsRNA resulted in apoptosis. Thus, CIV ORF 193R is the first iap gene identified in an iridovirus, which encodes a functional IAP protein.

Antiviral activity of lauryl gallate against animal viruses.

Antiviral compounds are needed in the control of many animal and human diseases. We analysed the effect of the antitumoural drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (herpes simplex and vaccinia) and RNA (influenza, porcine transmissible gastroenteritis and Sindbis) viruses, paying attention to its effect on the viability of the corresponding host cells. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 microM), reducing the titres 3->5 log units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5-8 h post-infection. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug; however, the early viral protein synthesis and the virus-mediated increase of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells. Furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. Lauryl gallate is a powerful antiviral agent against several pathogens of clinical and veterinary importance. The overall results indicate that a cellular factor or function might be the target of the antiviral action of alkyl gallates.

Role of the proximal enhancer of the major immediate-early promoter in human cytomegalovirus replication.

The human cytomegalovirus (CMV) enhancer has a distal component (positions -550 to -300) and a proximal component (-300 to -39) relative to the transcription start site (+1) of the major immediate-early (MIE) promoter. Without the distal enhancer, human CMV replicates slower and has a small-plaque phenotype. We determined the sequence requirements of the proximal enhancer by making 5'-end deletions to positions -223, -173, -116, -67, and -39. Even though recombinant virus with the proximal enhancer deleted to -39 has the minimal TATA box-containing MIE promoter element, it cannot replicate independently in human fibroblast cells. Recombinant virus with a deletion to -67 has an Sp-1 transcription factor binding site which may represent a minimal enhancer element for recombinant virus replication in human fibroblast cells. Although recombinant virus with a deletion to -223 replicates to titers at least 100-fold less than that of the wild-type virus, it replicates to titers 8-fold higher than that of recombinant virus with a deletion to -173 and 20-fold higher than that of virus with a deletion to -67. Recombinant virus with a deletion to -173 replicates more efficiently than that with a deletion to -116. There was a direct correlation between the level of infectious virus replication and time after infection, amount of MIE gene transcription, MIE and early viral protein synthesis, and viral DNA synthesis. The extent of the proximal enhancer determines the efficiency of viral replication.

Cocaine Potentiates the Switch between Latency and Replication of Epstein–Barr Virus in Raji Cells

This paper shows that cocaine amplifies Epstein–Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca2+ mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein–Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.

Fc receptors do not mediate african swine fever virus replication in macrophages

Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

Penetration of cells by herpes simplex virus does not require a low pH-dependent endocytic pathway

Agents that perturb endocytosis or that alter the pH of endosomes were shown to have little or no effect on plaque formation by herpes simplex virus (HSV), whereas plaque formation by vesicular stomatitis virus was inhibited as expected. A number of agents were tested for their ability to inhibit early events in HSV infection. Amantadine, chloroquine and trifluoperazine, whose actions are known to alter the endocytic pathway, showed no selective inhibitory effects on early events in HSV infection. Wheat germ agglutinin and heparin, known inhibitors of HSV infection, blocked the adsorption of virus to cells, as expected. Succinylated concanavalin A blocked plaque formation without inhibiting virus adsorption but could enhance the elution of bound virus. To a greater or lesser extent, succinylated concanavalin A, dithiothreitol, colchicine, monensin and cytochalasin B all inhibited or reduced the rate of events subsequent to adsorption and prior to early viral protein synthesis. Evidence is presented to suggest that each of these agents has a different mode of action. On the basis of these results and others, we conclude that endocytosis is probably not required for infection by HSV (at least not the low pH-dependent endocytic pathway) and that events occurring at the cell surface trigger virion-cell fusion leading to infection.

Saturable binding sites mediate the entry of African swine fever virus into VERO cells

Binding experiments of 3H-labeled African swine fever virus to susceptible VERO cells have shown the presence of saturable binding sites for African swine fever virus on the plasma membrane. The Scatchard analysis of the binding data at equilibrium indicates the existence of about 10 4 cellular receptor sites per cell with a dissociation constant ( K d ) of 70 p M. Virus entry into VERO cells is mediated by a saturable component, since tritiated African swine fever virus saturable binding and uptake were competed by the same amounts of unlabeled virus. Similarly, early viral protein synthesis and virus production were inhibited by concentrations of uv-inactivated virus that competed virus attachment to saturable binding sites, suggesting that specific receptors mediate the entry of African swine fever virus particles that initiate a productive infection in VERO cells. African swine fever virus binding to virus-resistant L cells was not mediated by saturable binding sites. As a result of the nonsaturable interaction the virus was not able to enter L cells and neither early viral protein synthesis nor viral DNA synthesis was detected, indicating that the absence of specific receptors for African swine fever virus is a factor that determines the resistance of L cells to the infection.

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase.

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.

Induction of cellular thymidine kinase occurs at the mRNA level.

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

Induction of cellular thymidine kinase occurs at the mRNA level.

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

The kinetics of synthesis of early viral proteins in KB cells infected with wild-type and transformation-defective host-range mutants of human adenovirus type 5.

We have studied the kinetics of early adenovirus type 5 (Ad5) protein synthesis during lytic infection of KB cells by wild-type (wt) and transformation-defective host-range (hr) mutants. Proteins encoded within four early regions were studied: early region 1A (E1A: 1.5 to 4.5 map units, mu), E1B (4.5 to 11.2 mu), E2A (61.6 to 74.9 mu), and E4 (91.4 to 99.1 mu). Synthesis of E1A products, the first to appear during wt lytic infection, was detectable within 2 h after injection, reached a peak within the next hour, then declined to very low levels by 7 h post-infection. Synthesis of E2 and E4 proteins began at about 3 h post-infection, was maximal by 6 h and thereafter declined sharply. The E1B 19K and 58K proteins were first detected around 3 h post-infection and, after reaching maximal levels of expression by 8 h, declined to lower levels by 12 h post-infection. Infections with the E1A mutant hr3 were characterized by greatly depressed levels of early expression of E1B, E2 and E4 polypeptides but protein synthesis from these regions appeared to recover at late times. The pattern of expression exhibited by the E1B mutant hr6 revealed delayed and reduced levels of expression of E1B, E2 and E4 protein synthesis but increased levels of E1A protein synthesis. These results are consistent with the reported role of E1A gene products in the activation of early gene expression and, in addition, suggest that a function encoded in E1B may also influence the expression of Ad5 early genes at early and late times.

The interaction of polyoma virus with F9 embryonal carcinoma cells and chemically induced differentiated progeny: fate of the viral DNA and expression of viral antigens.

Murine embryonal carcinoma (EC) cells fail to express viral T antigen following infection with polyoma (Py) virus, while some differentiated derivatives are fully susceptible to viral infection. The block to expression in EC cells apparently occurs after adsorption and penetration but prior to the synthesis of early viral proteins. The F9 EC cell line was employed to further investigate the failure of Py gene expression in EC cells as well as the viral susceptibility of certain differentiated cell types. F9 cultures treated with retinoic acid (RA) or RA in combination with dibutyryl cAMP (dBcAMP) are quantitatively induced to differentiate to endodermal cell types. When the fate of the viral DNA was examined in F9 EC cells, free viral DNA was observed early after infection but was eventually lost with continued cell growth. Cultures induced to differentiate in the presence of RA demonstrated limited viral susceptibility which increased with prolonged RA exposure. Polyoma sensitivity did not directly parallel basement membrane antigen production, a marker used to distinguish this differentiated endodermal cell type. Viral gene expression could be obtained if Py DNA was introduced into the cells prior to RA induction. In contrast, the endodermal progeny derived from a dual treatment with RA plus dBcAMP appeared highly refractory to Py infection.

Actin filament bundles in vaccinia virus infected fibroblasts.

When monolayer cultures of mouse 3T3 cells were infected with vaccinia virus, a rapid decrease of microfilament bundles was observed. The major disappearance of the cellular microfilaments occurred within the first hour after vaccinia virus infection, which is the period of uncoating of the virus core and early viral protein synthesis. The microfilament bundles of infected cells were significantly preserved in the presence of puromycin which inhibits viral protein synthesis.