Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.     

Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.     

Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a single (virus-encoded?) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381–3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.     

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography

Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.     

Effects of fatty acids on lipid synthesis and viral RNA replication in poliovirus-infected cells.

Animal viruses profoundly modify the metabolism of lipids, the synthesis of new membranes, and membrane traffic. These alterations are related to the replication of viral genomes. Addition of oleic acid from the beginning of poliovirus infection inhibits the appearance of virus polypeptides at concentrations that do not affect translation in mock-infected HeLa cells. This inhibition is due to the blockade of viral RNA synthesis. Membranes made in poliovirus-infected cells in the presence of oleic acid differ in their buoyant density from control membranes. These results suggest that the incorporation of oleic acid into membranes leads to increased membrane fluidity and decreased buoyant density, making these membranes nonfunctional for poliovirus RNA replication.     

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.     

3-Methylquercetin is a potent and selective inhibitor of poliovirus RNA synthesis

3-Methylquercetin (3MQ) is a natural compound isolated from Euphorbia grantii that selectively inhibits poliovirus replication, but has no effect on encephalomyocarditis virus. When the compound is present from the beginning of infection, the bulk of viral protein synthesis is prevented, but the shut-off of host protein synthesis still occurs. Addition of 3MQ 3 hr after infection has a slight effect on viral protein synthesis, suggesting that this compound blocks a step of viral replication different from translation. Indeed, poliovirus RNA synthesis is potently blocked by 3MQ, i.e., 50% inhibition at 2 micrograms/ml (6.3 X 10(-6) M). No effect on encephalomyocarditis, nor on cellular RNA synthesis is observed even at 20 micrograms/ml. The inhibitory effect of 3MQ is reversible, since cells treated with this compound from the beginning of infection start to synthesize viral RNA and proteins when the compound is removed. Strikingly, other natural compounds structurally related to 3-methylquercetin such as quercetin, naringenin, naringin, morin, catechin, kaempferol, myricetin, phloretin, phlorizdin, and rutin do not block poliovirus replication.     

Structure of Sm25, an antigenic integral membrane glycoprotein of adult Schistosoma mansoni

Sm25 is the principal antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental membranes of adult Schistosoma mansoni. The full-length amino acid sequence of this protein has been deduced from the sequence of two cDNAs, one isolated by screening a cDNA library and the other, including the 5' end of the gene, amplified directly from adult worm RNA using the polymerase chain reaction. The predicted sequence represents a nascent polypeptide of Mr 21,500. Following cleavage of a predicted signal sequence, the Mr of the resulting polypeptide is 17,600. The polypeptide contains 2 potential sites for N-linked glycosylation and a hydrophobic domain at the C-terminus that could facilitate membrane association. Analysis of the mature gene product confirmed that Sm25 is an N-glycosylated integral membrane protein and that the Mr of the deglycosylated polypeptide is between 15,000 and 20,000.     

Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells.

As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.     

In vivo regulation of the poliovirus RNA polymerase.

The viral polypeptide isolated with the poliovirus replication complex appears to be an essential component of the poliovirus RNA-dependent RNA polymerase. This viral polypeptide is synthesized early in the infectious cycle, is directly proportional to viral RNA synthesis throughout most of the replicative cycle, and is functionally stable. However, the level of functioning polymerase molecules within the infected cell represents only a small fraction of the potential polymerase polypeptides synthesized during poliovirus replication. This disparity indicates that this viral polypeptide, while necessary, is not sufficient for viral RNA synthesis. We suggest that an additional factor must restrict the level of functioning viral polymerases within the infected cell.     

Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis

The polypeptide 3CD of many poliovirus strains can be cleaved at two different amino acid pairs. The viral proteinase 3C and the viral polymerase 3D result from cleavage at a Gln-Gly pair by proteinase 3C, whereas cleavage at a Tyr-Gly pair by proteinase 2A yields the alternative products 3C' and 3D'. Specific mutations were introduced into the 3C'/3D' cleavage site in an infectious cDNA clone of poliovirus type 1 (Mahoney) by oligonucleotide-directed mutagenesis in order to investigate the role of 3C' and 3D' in viral proliferation and to obtain information about the cleavage specificity of 2Apro. Substitution of a threonine residue by an alanine residue at position -2 (P2) of this cleavage site abolished cleavage, whereas substitution of a tyrosine residue by a phenylalanine residue at amino acid position -1 (P1) of the cleavage site did not influence processing. Both mutated cDNA clones produced infectious viruses (T147A and Y148F) on transfection. The phenotypes of the mutant viruses were similar to that of the parental strain. We conclude that (i) 3C' and 3D' are not essential for virus replication, (ii) a Phe-Gly pair at the cleavage site can be cleaved by 2Apro, and (iii) a threonine residue in the P2 position of the cleavage site may be important in substrate recognition by 2Apro.     

Modulation of the expression of poliovirus proteins in reticulocyte lysates

The translation of poliovirus RNA into specific viral proteins in mRNA-dependent reticulocyte lysates (MDLs) was found to be highly dependent on individual lysate preparations. Under optimal conditions, the first polypeptide detected was always P3-1b (formerly NCVP 1b), the product of the 3' portion of the poliovirus genome; the formation of P1-1a (formerly NCVP 1a) followed as shown by time-course and pulse-chase experiments. However, some lysates synthesized little or no P1-1a despite their ability to synthesize P3-1b and to translate normally other cellular and viral mRNAs. When an MDL competent in synthesizing P1-1a was diluted ca. twofold, while maintaining optimal concentrations of salts, tRNA, DTT, creatine phosphate, and amino acids, P1-1a formation was virtually eliminated, while the synthesis of P3-1b, presumably as a consequence of a more downstream initiation, was maintained. The synthesis of P1-1a in a diluted MDL was restored, and P3-1b synthesis suppressed, by the addition of a S10 fraction prepared from uninfected or virus-infected HeLa cells. Nuclease treatment and dialysis of the S10 fraction did not inhibit its activity. These findings indicate that individual MDLs either possess limiting quantities of, or occasionally are deficient in, a factor(s) that promotes the utilization of the presumed 5' proximal initiation site (the AUG at nucleotide position 781-783) and that a homologous factor(s) exists in HeLa cells. The implication of these findings for the strategy of poliovirus replication is discussed.     

Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A.

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.     

The E5 oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasma membranes

The E5 oncoprotein of BPV-1 is a 44 amino acid, hydrophobic polypeptide which is present in membrane fractions of transformed cell homogenates. To define the specific cellular membrane compartments with which E5 associates, immunofluorescence and immunoelectron microscopy were performed using an affinity-purified antibody specific for the E5 carboxyl-terminus. Two cell systems which overexpressed the E5 protein were analyzed: (1) Balb/c 3T3 mouse cells transformed by a BPV-1 cDNA expressing the E2-E5 ORFs, and (2) Sf9 insect cells infected with recombinant baculovirus expressing the E5 ORF. In both cell types, E5 protein was found in the membranes of the Golgi apparatus with its carboxyl-terminus oriented intraluminally. In the Sf9 cells, which synthesize much greater quantities of viral protein, E5 protein was observed additionally in plasma membranes with the carboxyl-terminus facing extracellularly. The concentration of E5 protein within the Golgi and plasma membranes suggests several potential mechanisms for inducing cellular transformation.     

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3′UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3′UTR. NS5A can still regulate viral RNA synthesis through binding to 3′UTR when binding to NS5B is not available.     

Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus

The complete sequence of Punta Toro virus (Phlebovirus, Bunyaviridae) middle size (M), RNA has been determined. The RNA is 4330 nucleotides long (mol wt 1.46 X 10(6), base composition: 26.7% A, 33.6% U, 18.5% G, 21.2% C) and has 3'- and 5'-terminal sequences that, depending on the arrangement, are complementary for some 15 residues. The viral RNA codes in its viral-complementary sequence for a single primary gene product (the viral glycoprotein precursor) that is comprised of 1313 amino acids (146,376 Da) and is abundant in cysteine residues but has few potential asparagine-linked glycosylation sites. The 5'-noncoding region of the Punta Toro M viral-complementary RNA is short (16 nucleotides); the 3'-noncoding sequence is much longer (372 nucleotides). The latter is rich in short stretches of adenylate residues, like the 3'-noncoding regions of the Punta Toro S mRNA species (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306). No other large open reading frame has been identified in either the viral, or viral-complementary, M RNA sequences. Limited amino-terminal sequence analyses of the two viral glycoproteins have indicated the gene order and potential cleavage sites in the glycoprotein precursor. The data suggest the existence of a 30 X 10(3)-Da polypeptide (designated NSM) in the glycoprotein precursor that precedes the G1 protein (i.e., gene product order: NSM-G1-G2). Examination of the sequence of the Punta Toro M gene product reveals the presence of multiple hydrophobic sequences including a 19-amino acid, carboxy-proximal, hydrophobic region (G2). This hydrophobic sequence is followed by a 13-amino acid-terminal sequence rich in charged amino acids. The size and constitution of the carboxy-terminal region is consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. Other regions of the glycoprotein precursor contain sequences of amino acids with a predominantly hydrophobic character (23, 50, and 20 amino acids in length). Their functions are unknown. The amino terminus of the G1 protein is located near the end of the 23-amino acid-long hydrophobic sequence of the presumptive precursor, the hydrophobic 50-amino acid sequence lies within G1, and the amino terminus of G2 is located in the middle of the 20-amino acid-long hydrophobic sequence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete nucleotide sequence of the Toledo isolate of turnip ringspot virus

The complete genomic sequence of the Toledo isolate of the comovirus, turnip ringspot virus (TuRSV), was found to consist of 2 polyadenylated RNAs. RNA 1 is 6082 nucleotides long and encodes a single predicted polypeptide of 1860 amino acids. The predicted RNA 1 polyprotein contains the polypeptides for viral replication and proteolytic processing. RNA 2, that is 3985 nucleotides long, codes for a single predicted 1095 amino acid polypeptide containing the movement and coat proteins. Phylogenetic analysis indicates that TuRSV is most closely related to radish mosaic virus, and these crucifer-infecting pathogens form a distinct clade within the comoviruses.