Virus-specific Protein Synthesis Research Articles

Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

The innate immune response is the first line of defense against most viral infections. Its activation promotes cell signaling, which reduces virus replication in infected cells and leads to induction of the antiviral state in yet-uninfected cells. This inhibition of virus replication is a result of the activation of a very broad spectrum of specific cellular genes, with each of their products usually making a small but detectable contribution to the overall antiviral state. The lack of a strong, dominant function for each gene product and the ability of many viruses to interfere with the development of the antiviral response strongly complicate identification of the antiviral activity of the activated individual cellular genes. However, we have previously developed and applied a new experimental system which allows us to define a critical function of some members of the poly(ADP-ribose) polymerase (PARP) family in clearance of Venezuelan equine encephalitis virus mutants from infected cells. In this new study, we demonstrate that PARP7, PARP10, and the long isoform of PARP12 (PARP12L) function as important and very potent regulators of cellular translation and virus replication. The translation inhibition and antiviral effect of PARP12L appear to be mediated by more than one protein function and are a result of its direct binding to polysomes, complex formation with cellular RNAs (which is determined by both putative RNA-binding and PARP domains), and catalytic activity. IMPORTANCE The results of this study demonstrate that interferon-stimulated gene products PARP7, PARP10, and PARP12L are potent inhibitors of the replication of Venezuelan equine encephalitis virus and other alphaviruses. The inhibitory functions are determined by more than a single mechanism, and one of them is based on the ability of these proteins to regulate cellular translation. Interference with the cellular translational machinery depends on the integrity of both the amino-terminal domain, containing a number of putative RNA-binding motifs, and the catalytic function of the carboxy-terminal PARP domain. The PARP-induced changes in translation efficiency appear to have a more potent effect on the synthesis of virus-specific proteins than on that of cellular proteins, thus making PARP-specific translational downregulation an important contributor to the overall development of the antiviral response.

Application of allele-specific RNAi in hepatitis B virus lamivudine resistance

Understanding the consequences of mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus (HBV) genome on replication is critical for treating chronic hepatitis B with lamivudine. Allele-specific gene silencing by RNAi (allele-specific RNAi: ASP-RNAi) is an advanced application of RNAi techniques. Use of this strategy as a means for specifically inhibiting an allele expression of interest suggested that it can specifically suppress the expression of alleles causing disease without inhibiting the expression of corresponding wild-type alleles. However, no studies have used ASP-RNAi to address the issue of HBV lamivudine resistance. In this study, we applied ASP-RNAi into two long-term eukaryotic cell lines of full-length HBV containing either lamivudine-resistant mutants (HBV-YIDD) or wild type (HBV-WT) which we generated in previously. The designed siRNAs were also used in this eukaryotic expression system together with lamivudine. ELISA and real-time PCR were performed to monitor virus-specific protein synthesis and viral DNA replication. The results showed that the base substitutions conferring marked ASP-RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15 and 19 of the sense strand of siRNAs which were different from the most sensitive positions of this application in eukaryotes. In addition, siRNA-lamivudine combinations did not possess the prominent anti-HBV activity we expected because of some unknown mechanisms. These findings recapitulated many of the features of ASP-RNAi in hepadnaviruses which provided a new insight into the development of a potent strategy against HBV drug resistance.

Blockade of the Poliovirus-Induced Cytopathic Effect in Neural Cells by Monoclonal Antibody against Poliovirus or the Human Poliovirus Receptor

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2Apro, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2Apro was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2Apro is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.

In vitro anti-influenza virus activity of the pavine alkaloid (-)-thalimonine isolated from Thalictrum simplex L.

The pavine alkaloid (-)-thalimonine (Thl), isolated from the Mongolian plant Thalictrum simplex inhibited markedly the reproduction of influenza virus A/Germany/27, str. Weybridge (H7N7) and A/Germany/34, str. Rostock (H7N1) in cell cultures of chicken embryo fibroblasts. In a number of assays at a non-toxic concentration range of 0.1-6.4 microM the alkaloid inhibited viral reproduction in a selective and specific way (selectivity index = 640, 106.6, respectively). Expression of viral glycoproteins haemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) on the surface of infected cells, virus-induced cytopathic effect, infectious virus yields, HA production and virus-specific protein synthesis were all reduced. The inhibition was dose-related and depended on virus inoculum. The time of addition experiments indicated that viral reproduction was markedly inhibited when Thl was added at 4-5 h of infection. No inactivating effect on extracellular virus was found.

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells.

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.

Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.

Inhibition of influenza A virus reproduction by a ribozyme targeted against PB1 mRNA

A ribozyme gene directed at a specific cleavage of mRNA coding for PB1 protein, a component of RNA-dependent RNA-polymerase of influenza A virus, was constructed. The avian adenovirus CELO virus-associated RNA (VA RNA CELO) promoter and human cytomegalovirus (CMV) promoter were used for the permanent expression of the ribozyme in cell lines. The cells were infected with influenza A virus strains A/Singapore/1/57 and A/WSN/33, and the suppression of the virus reproduction and virus-specific protein synthesis was measured. The maximal level of the inhibition of virus reproduction as compared to the reproduction in non-transformed cells was 93.5%. Defective recombinant adenoviruses were constructed carrying the genes of functional and non-functional ribozymes under the control of human cytomegalovirus (CMV) promoter. The reproduction of A/WSN/33 virus in CV-1 cells preinfected with recombinant adenoviruses was shown to be suppressed.

Inhibition of influenza A virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin.

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.

Modification of cytoskeleton and prosome networks in relation to protein synthesis in influenza A virus-infected LLC-MK2 cells

Mofications of the cytoskeleton and protein synthesis were investigated in LC-MK2 cells during infection by FPV/Ulster 73, an avian strain of influenza A virus. During infectio, the cytoskeleton and the prosome networks undergo a dramatic reorganization, which seems to be at least temporally differentiated for each cytoskeletal system, i.e. microfilaments (MFs), microtubules (MTs), intermediate filaments (IFs). In order to evaluate the role of the three different cytoskeletal networks during FPV/Ulster infection, studies were carried out on cellular and virus-specific protein synthesis and viral production, using drugs which selectively affect individual cytoskeletal systems. Our data show that the perturbation of the IF system, but not that of the MFs or MTs, seems to have a strong inhibitory effect on virus production and cellular and viral protein synthesis. Furthermore, the dynamics of IFs and prosomes were investigated during viral infection and, at no time, dissociation of the prosome and IF networks was observed. Taken together, these results strongly support the idea that the interactions between the protein synthesis machinery, the cytoskeleton, and the prosomes are all affected by viral infection in a partially coordinated manner.

Antiviral Activity of an Aqueous Extract of the CyanobacteriumMicrocystis aeruginosa

The crude aqueous extract of a field collected strain of Microcystis aeruginosa (Kütz) showed antiviral activity against influenza A virus. The concentration for 50% inhibition of virus replication in MDCK cells was 11 μg dry extract/mL. The virus specific protein synthesis decreased if the extract was present over the whole time of replication. Furthermore protease inhibitory activity was estimated for the crude aqueous extract and subextracts. © 1997 by John Wiley & Sons, Ltd.

Entry and Release of Measles Virus Are Polarized in Epithelial Cells

The initial site of virus replication during measles infection is in the epithelial cells of the respiratory tract. We have investigated measles virus infection of two types of polarized epithelial cells to determine if entry and/or release of the virus is confined to either the apical or the basolateral plasma membrane. The Caco-2 line of human intestinal epithelial cells and the polarized Vero C1008 monkey kidney cell line were grown on permeable supports and inoculated either through the apical or basolateral surfaces. Cells exposed to virus in the apical medium showed high levels of synthesis of virus-specific proteins, whereas no synthesis of viral proteins was detected in cells inoculated at the basolateral surface. Virus titers derived from apically infected cells were found to be about 1000-fold greater than titers derived from cells infected at the basolateral surface. Indirect immunofluorescence results also demonstrated that expression of measles viral antigens occurs at high levels only when input virions are inoculated at the apical surface. To investigate the localization of CD46 and moesin, which are receptors for measles virus, Caco-2 cells were incubated with monoclonal antibodies against CD46 or moesin followed by 125I-labeled anti-mouse Ig.The results indicate that CD46 is expressed preferentially on the apical membranes while moesin appears to be present at similar levels on both surfaces. Release of the virus was also examined and found to be polarized as well Virus was released into the apical medium at up to 1000-fold higher titers than virus released into the basolateral medium. These results demonstrate that in two epithelial cell types measles virus preferentially enters and is released from epithelial cells in a polarized fashion through the apical plasma membrane.

Synthesis and assembly of hepatitis A virus-specific proteins in BS-C-1 cells.

To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35S]methionine and L-[35S]cysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VP0, VP2, and VP3; procapsids contained VP1, VP0, and VP3; and pentamers contained PX, VP0, and VP3, as previously shown by immunoblotting (D.A. Anderson and B.C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1- and VP2-reactive sequences and sedimenting slightly more slowly than 14S pentamers, which may represent intact P12A assembled into pentamers as has been reported for the P1 of some other picornaviruses (S. McGregor and R. R. Rueckert, J. Virol. 21:548-553, 1977). The results of this study suggest that cleavage of the hepatitis A virus polyprotein to produce pentamers is protracted (though not rate limiting) early in infection, while the assembly of pentamers into higher structures is a rapid process once sufficient viral RNA is produced for encapsidation.

The Receptor-Destroying Enzyme of Influenza C Virus Is Required for Entry into Target Cells

The hemagglutinin-esterase (HE) protein of influenza C viruses possesses an acetylesterase activity, which appears essential for replication, as determined by reduced infectivity after inhibition of the viral enzyme [Vlasak et al., J. Virol. 63, 2056-2062 (1989)]. Analysis revealed the absence of virus-specific RNA and protein synthesis in infected cells after inhibition of the receptor-destroying enzyme. In addition, hemolytic activity was reduced after incubation of influenza C/JJ/50 virus with diisopropyl-fluorophosphate or 3,4-dichloro-isocoumarin. Further analysis revealed that inhibition of hemolysis depends on virus and erythrocyte concentrations. It is suggested that an active receptor-destroying enzyme is required for entry of influenza C virus into target cells at a step prior to fusion of the vital and cellular membrane. Our data indicate that cleavage of receptors bound to the HE protein is a prerequisite for the low pH-triggered conformational change required for fusion.

Anti-influenza virus activity of the compound LY253963

The compound LY253963 (1,3,4-thiadiazol-2-ylcyanamide) inhibited the in vitro replication of representative influenza A and B viruses in Madin-Darby canine kidney (MDCK) cells at concentrations of 1–3.2 μg/ml. The yield of an influenza A (H3N2) virus in primary rhesus monkey kidney (RMK) cells was inhibited at 0.1–0.3 μg/ml. However, similar concentrations were inhibitory for the growth of uninfected MCDK or RMK cells. Combination drug studies generally found indifferent interactions between LY253963 and ribavirin or rimantadine. In timing of additional studies, hemagglutinin expression was inhibited to the greatest extent when LY253963 exposure was begun at least 8 h before viral infection, which suggested either slow uptake or intracellular metabolism of LY253963 to an active form. Virus-specific protein synthesis was inhibited to a greater extent by ribavirin 10 μg/ml or rimantadine 1 μg/ml than by LY253963 10 μg/ml. No drug-resistant mutants were detected during serial passage of an influenza A (H3N2) virus in the presence of LY253963 1–16 μg/ml. In summary, we found that LY253963 inhibited influenza A and B virus replication in several cell types, but that it was associated with cytostatic effects at low concentrations. These studies failed to identify a selective anti-influenza action.

Macromolecular synthesis in varicella-zoster virus-infected epithelial and fibroblast cells

Replication of varicella-zoster virus (VZV) was studied in a range of human cells (Chang conjunctival, HEp2 and Vero) and compared with that in HEL cells. Evidence for virus-specific protein and nucleic acid synthesis was obtained from each of these cell lines. In some cell lines, virus proteins were clearly visible by polyacrylamide gel electrophoresis both with and without immunoprecipitation. Multiple passage of infected cells enhanced both virus-specific protein synthesis and shutoff of host protein synthesis. No qualitative differences in the patterns of VZV-specific protein synthesis were detected. Approximately 2000 copies of the VZV genome were detected per infected HEL cell.

A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells

To enhance the penetration of oligonucleotide (‘oligo’) into cells, the oligo was combined with the hydrophobic undecyl residue. Using the ‘DNA-synthesator’, we synthesized oligo, complementary to the loop-forming site of the RNA, encoding polymerase 3 of the influenza virus (type A), and combined it with the undecyl residue added to the 5' terminal phosphate group. It was found that the modified oligo effectively suppresses the influenza A/PR8/34 (H1N1) virus reproduction and inhibits the synthesis of virus-specific proteins in MDCK cells. Under the same conditions, the non-modified antisense oligo and modified nonsense oligo did not affect the virus development.

Antisense oligonucleotides modified at 5'-end by fatty radicals effectively inhibit reproduction of influenza virus

To enhance penetration of oligonucleotides ("oligos") into cells it was suggested that they should be chemically modified by fatty radicals at 5'-end. Two modified by undecanol at 5'-end phosphate group oligos, namely, oligo complementary to the protein binding sites, located at the influenza virus polymerases encoding RNA, and oligo complementary to the polyadenylation signal of the polymerase 3 encoding RNA were synthesized using DNA-synthetisator. These modified oligos effectively suppressed the influenza A/PR8/34 virus reproduction and inhibited the synthesis of virus-specific proteins in MDCK cells. The non-modified antisense oligos and modified nonsense oligos did not affect the virus development under the same conditions.

Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

The deletion of 41 proximal nucleotides reverts a poliovirus mutant containing a temperature-sensitive lesion in the 5' noncoding region of genomic RNA

We generated a number of small deletions and insertions in the 5' noncoding region of an infectious cDNA copy of the poliovirus RNA genome. Transfection of these mutated cDNAs into COS-1 cells produced the following phenotypic categories: (i) wild-type mutations, (ii) lethal mutations, (iii) mutations exhibiting slow growth or low-titer properties, and (iv) temperature-sensitive (ts) mutations. The deletion of nucleotides 221 to 224 produced a ts virus, 220D1. Mutant 220D1 was found to have a dramatic reduction in growth, virus-specific protein and RNA synthesis, and the shutoff of host cell protein synthesis at 37 or 39 degrees C compared with 33 degrees C. Temperature shift experiments showed that the mutant viral RNA is not an effective template for protein or RNA synthesis at 39 degrees C and suggested a decreased stability of the 220D1 RNA at 39 degrees C. Selection for a non-ts revertant of 220D1 yielded the virus R2, which was no longer ts for growth or viral protein and RNA synthesis. Sequencing the 5' noncoding region of the genomic RNA from R2 revealed the deletion of 41 proximal nucleotides for an overall deletion of nucleotides 184 to 228. These data suggest that the deleted sequences are nonessential to the poliovirus life cycle during growth in HeLa cells. According to computer-predicted RNA secondary structures of the 5' noncoding region of poliovirus RNA, the R2 revertant virus has deleted an entire predicted stem-loop structure.

Influence of bonaphthone on the synthesis of virus-specific proteins of herpes simplex virus, type 1

Influence of bonaphthone on the synthesis of virus-specific proteins of herpes simplex virus, type 1