Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular... more Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle ofthe virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.
Page 1. 482 ti on^.^^ In the radical ion mechanism, the bridging ligand is first reduced to a rad... more Page 1. 482 ti on^.^^ In the radical ion mechanism, the bridging ligand is first reduced to a radical which subsequently reduces the oxidizing metal center to which it is coor-dinated. In the resonance transfer or exchange mech ...
Biochemical and Biophysical Research Communications, Apr 26, 2002
Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packa... more Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5 0 -ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp a p b -p a A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction. Ó
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1987
The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-as... more The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-associated RNA polymerase L protein, transcribes the genome ribonucleoprotein template in vitro. It contains an acidic N-terminal domain and two distinct domains at the C-terminal end that are involved in binding to the polymerase protein and the template RNA enwrapped with the nucleocapsid protein. In the present study, the portions of the NS gene that encode the N- and C-terminal domains of the protein were cloned in pGEM vectors and expressed by in vitro transcription and translation. It was shown that two polypeptides obtained by translation of the encoded mRNAs support RNA synthesis in vitro in a reconstitution reaction when they are added together in trans. Moreover, the N-terminal domain can be functionally substituted by structurally similar polypeptides.
Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent ... more Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-ζ) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-ζ has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-ζ has only minor activity. Selective inhibition of PKC-ζ activity within CV1 cells eliminated permissiveness to CDV replication, indicating anin vivorole for PKC-ζ in the virus replication cycle. The broad tissue expression of PKC-ζ parallels the pantropic nature of CDV infections, suggesting that PKC-ζ activity is a determinant of cellular permissiveness to CDV replication.
The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (... more The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.
Under appropriate reaction conditions in vitro, four different defective-interfering particles of... more Under appropriate reaction conditions in vitro, four different defective-interfering particles of vesicular stomatitis virus have been shown to synthesize the full-length complement of their RNAs. The reaction involved preinitiation of the core particles with ATP and CTP, followed by RNA chain elongation in the presence of the beta , gamma -imido analogue of ATP, AdoPP[NH]P, and the three normal ribonucleoside triphosphates. By hybridization of the in vitro synthesized plus strand with the standard genome RNA followed by RNase treatment of the heteroduplexes, we have shown that the RNA of a defective-interfering particle derived from the 3' end of the genome RNA has evolved by an internal deletion of the standard genome.
The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis vi... more The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis virus [New Jersey serotype, VSV(NJ)] was derived from a cDNA clone and mRNA. The mRNA is 758 nucleotides long (excluding polyadenylic acid) and encodes a protein of 229 amino acids. The predicted amino acid sequence was compared with that of the corresponding protein of Indiana serotype [VSV(IND)] and a fish rhabdovirus, spring viremia of carp virus (SVCV). An amino acid identity of 62% was found between the M proteins of VSV(NJ) and VSV(IND) while only 24% was present between VSV(NJ) and SVCV. A highly basic NH2-terminal domain followed by a proline-proline-X-tyrosine sequence was present in all the three M polypeptides. Except for the L gene sequence, the complete nucleotide sequence of the four genes of VSV(NJ) are now known. The comparison of the amino acid sequences between the Indiana and New Jersey serotypes demonstrates a high degree of homology between these genes except for the phosphoprotein gene, NS.
Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, l... more Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infectionrelated damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility
NF-B is known to exert its antiviral innate immune response via the IFN--induced Janus kinase/sig... more NF-B is known to exert its antiviral innate immune response via the IFN--induced Janus kinase/signal transducers and activators of transcription pathway. However, our current studies have demonstrated that activated NF-B is capable of directly establishing an antiviral state independent of IFN or secreted soluble factor(s) against two highly pathogenic respiratory RNA viruses. Human parainfluenza virus type 3, a mildly cytopathic virus that induced NF-B very early during infection was converted to a virulent virus after NF-B inhibition. In contrast, a highly cytopathic virus, human respiratory syncytial virus that induced NF-B late during infection, was converted to a mildly cytopathic virus after NF-B induction before virus replication. This interconversion of cytopathic phenotypes of viruses after NF-B modulation was further shown to be independent of IFN and soluble secreted factors(s). Moreover, tumor necrosis factor (TNF-) and IL-1 elicited an antiviral response, which was NF-B-dependent. Thus, NF-B induction directly confers an essential innate antiviral response against human parainfluenza virus type 3 and respiratory syncytial virus, which is independent of IFN-inducible factor(s).
The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-depe... more The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein.
The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, wer... more The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.
Proceedings of the National Academy of Sciences of the United States of America, 1995
Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for the... more Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.
Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomat... more Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomatitis virus transcribes RNA have revealed several new biological features of general interest. The mode of synthesis of the 5'-terminal cap structure of the mRNAs, the sequential transcription of the genes and the presence of a transcribed 'leader' RNA segment are properties which are either not shown by other viruses, or have not yet been described. These features are probably inter-related with the primary transcription process, which itself may be a useful mode/for future studies on mRNA biosynthesis in eukaryotic systems.
The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase... more The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.
Irradiation of purified influenza virus and vesicular stomatitis virus (VSV) with long-wavelength... more Irradiation of purified influenza virus and vesicular stomatitis virus (VSV) with long-wavelength UV light in the presence of 4'-substituted psoralens inactivated the virion-associated RNA polymerase activity. Inactivation was apparently due to psoralen modification of the viral genome RNAs, since cations that decrease psoralen binding to nucleic acids had a protective effect, and reconstitution of VSV RNA polymerase activity was inhibited by photoreaction of nucleoprotein cores but not by pretreatment of soluble fraction from dissociated virions. Partially inactivated viral particles synthesized reduced amounts of full-length RNA products in vitro without an increase in prematurely terminated transcripts. VSV leader RNA formation was relatively resistant to psoralen photoinactivation, and sequential transcription was maintained by photoreacted VSV. The all-or-none psoralen effect on virion-associated RNA polymerase activities may be due to a differential photosensitivity of promoter sites or to structural changes in modified viral genome RNAs that prevent formation of new mRNA chains.
Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular... more Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle ofthe virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.
Page 1. 482 ti on^.^^ In the radical ion mechanism, the bridging ligand is first reduced to a rad... more Page 1. 482 ti on^.^^ In the radical ion mechanism, the bridging ligand is first reduced to a radical which subsequently reduces the oxidizing metal center to which it is coor-dinated. In the resonance transfer or exchange mech ...
Biochemical and Biophysical Research Communications, Apr 26, 2002
Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packa... more Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5 0 -ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp a p b -p a A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction. Ó
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1987
The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-as... more The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-associated RNA polymerase L protein, transcribes the genome ribonucleoprotein template in vitro. It contains an acidic N-terminal domain and two distinct domains at the C-terminal end that are involved in binding to the polymerase protein and the template RNA enwrapped with the nucleocapsid protein. In the present study, the portions of the NS gene that encode the N- and C-terminal domains of the protein were cloned in pGEM vectors and expressed by in vitro transcription and translation. It was shown that two polypeptides obtained by translation of the encoded mRNAs support RNA synthesis in vitro in a reconstitution reaction when they are added together in trans. Moreover, the N-terminal domain can be functionally substituted by structurally similar polypeptides.
Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent ... more Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-ζ) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-ζ has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-ζ has only minor activity. Selective inhibition of PKC-ζ activity within CV1 cells eliminated permissiveness to CDV replication, indicating anin vivorole for PKC-ζ in the virus replication cycle. The broad tissue expression of PKC-ζ parallels the pantropic nature of CDV infections, suggesting that PKC-ζ activity is a determinant of cellular permissiveness to CDV replication.
The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (... more The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.
Under appropriate reaction conditions in vitro, four different defective-interfering particles of... more Under appropriate reaction conditions in vitro, four different defective-interfering particles of vesicular stomatitis virus have been shown to synthesize the full-length complement of their RNAs. The reaction involved preinitiation of the core particles with ATP and CTP, followed by RNA chain elongation in the presence of the beta , gamma -imido analogue of ATP, AdoPP[NH]P, and the three normal ribonucleoside triphosphates. By hybridization of the in vitro synthesized plus strand with the standard genome RNA followed by RNase treatment of the heteroduplexes, we have shown that the RNA of a defective-interfering particle derived from the 3' end of the genome RNA has evolved by an internal deletion of the standard genome.
The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis vi... more The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis virus [New Jersey serotype, VSV(NJ)] was derived from a cDNA clone and mRNA. The mRNA is 758 nucleotides long (excluding polyadenylic acid) and encodes a protein of 229 amino acids. The predicted amino acid sequence was compared with that of the corresponding protein of Indiana serotype [VSV(IND)] and a fish rhabdovirus, spring viremia of carp virus (SVCV). An amino acid identity of 62% was found between the M proteins of VSV(NJ) and VSV(IND) while only 24% was present between VSV(NJ) and SVCV. A highly basic NH2-terminal domain followed by a proline-proline-X-tyrosine sequence was present in all the three M polypeptides. Except for the L gene sequence, the complete nucleotide sequence of the four genes of VSV(NJ) are now known. The comparison of the amino acid sequences between the Indiana and New Jersey serotypes demonstrates a high degree of homology between these genes except for the phosphoprotein gene, NS.
Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, l... more Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infectionrelated damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility
NF-B is known to exert its antiviral innate immune response via the IFN--induced Janus kinase/sig... more NF-B is known to exert its antiviral innate immune response via the IFN--induced Janus kinase/signal transducers and activators of transcription pathway. However, our current studies have demonstrated that activated NF-B is capable of directly establishing an antiviral state independent of IFN or secreted soluble factor(s) against two highly pathogenic respiratory RNA viruses. Human parainfluenza virus type 3, a mildly cytopathic virus that induced NF-B very early during infection was converted to a virulent virus after NF-B inhibition. In contrast, a highly cytopathic virus, human respiratory syncytial virus that induced NF-B late during infection, was converted to a mildly cytopathic virus after NF-B induction before virus replication. This interconversion of cytopathic phenotypes of viruses after NF-B modulation was further shown to be independent of IFN and soluble secreted factors(s). Moreover, tumor necrosis factor (TNF-) and IL-1 elicited an antiviral response, which was NF-B-dependent. Thus, NF-B induction directly confers an essential innate antiviral response against human parainfluenza virus type 3 and respiratory syncytial virus, which is independent of IFN-inducible factor(s).
The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-depe... more The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein.
The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, wer... more The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.
Proceedings of the National Academy of Sciences of the United States of America, 1995
Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for the... more Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.
Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomat... more Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomatitis virus transcribes RNA have revealed several new biological features of general interest. The mode of synthesis of the 5'-terminal cap structure of the mRNAs, the sequential transcription of the genes and the presence of a transcribed 'leader' RNA segment are properties which are either not shown by other viruses, or have not yet been described. These features are probably inter-related with the primary transcription process, which itself may be a useful mode/for future studies on mRNA biosynthesis in eukaryotic systems.
The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase... more The smallest size class of mRNA (12S) synthesized in vitro by the virionassociated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.
Irradiation of purified influenza virus and vesicular stomatitis virus (VSV) with long-wavelength... more Irradiation of purified influenza virus and vesicular stomatitis virus (VSV) with long-wavelength UV light in the presence of 4'-substituted psoralens inactivated the virion-associated RNA polymerase activity. Inactivation was apparently due to psoralen modification of the viral genome RNAs, since cations that decrease psoralen binding to nucleic acids had a protective effect, and reconstitution of VSV RNA polymerase activity was inhibited by photoreaction of nucleoprotein cores but not by pretreatment of soluble fraction from dissociated virions. Partially inactivated viral particles synthesized reduced amounts of full-length RNA products in vitro without an increase in prematurely terminated transcripts. VSV leader RNA formation was relatively resistant to psoralen photoinactivation, and sequential transcription was maintained by photoreacted VSV. The all-or-none psoralen effect on virion-associated RNA polymerase activities may be due to a differential photosensitivity of promoter sites or to structural changes in modified viral genome RNAs that prevent formation of new mRNA chains.
Uploads
Papers by Amiya Banerjee