5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.     

Identification of a cis-acting replication element within the poliovirus coding region

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.     

Nodavirus RNA replication protein a induces membrane association of genomic RNA

Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (A(D692E)), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein A(D692E) stimulated RNA1 accumulation in wild-type cells and in xrn1(-) yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.     

Action of 3-methylquercetin on poliovirus RNA replication.

3-Methylquercetin is a natural flavone that powerfully blocks poliovirus replication. This compound inhibits selectively poliovirus RNA synthesis both in infected cells and in cell-free systems. Poliovirus double-stranded RNA (replicative forms) is still made in the presence of this inhibitor, whereas the synthesis of single-stranded RNA and the formation of replicative intermediates are drastically blocked.     

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.     

Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA.

A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed.     

Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.     

Self-catalyzed linkage of poliovirus terminal protein VPg to poliovirus RNA

The poliovirus terminal protein, VPg, was covalently linked to poliovirus RNA in a reaction that required synthetic VPg, Mg2+, and a replication intermediate synthesized in vitro. The VPg linkage reaction did not require the viral polymerase, host factor, or ribonucleoside triphosphates and was specific for template-linked minus-strand RNA synthesized on poliovirion RNA. The covalent nature of the bond between VPg and the RNA was demonstrated by the isolation of VPg-pUp from VPg-linked RNA. A model is proposed in which the tyrosine residue in VPg forms a phosphodiester bond with the 5'UMP in minus-strand RNA in a self-catalyzed transesterification reaction. It appears that either the RNA, VPg, or a combination of both forms the catalytic center for this reaction.     

Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal.

The mouse hepatitis virus (MHV) sequences required for replication of the JHM strain of MHV defective interfering (DI) RNA consist of three discontinuous genomic regions: about 0.47 kb from both terminal sequences and a 0.13-kb internal region present at about 0.9 kb from the 5' end of the DI genome. In this study, we investigated the role of the internal 0.13-kb region in MHV RNA replication. Overall sequences of the 0.13-kb regions from various MHV strains were similar to each other, with nucleotide substitutions in some strains; MHV-A59 was exceptional, with three nucleotide deletions. Computer-based secondary-structure analysis of the 0.13-kb region in the positive strand revealed that most of the MHV strains formed the same or a similar main stem-loop structure, whereas only MHV-A59 formed a smaller main stem-loop structure. The RNA secondary structures in the negative strands were much less uniform among the MHV strains. A series of DI RNAs that contained MHV-JHM-derived 5'- and 3'-terminal sequences plus internal 0.13-kb regions derived from various MHV strains were constructed. Most of these DI RNAs replicated in MHV-infected cells, except that MRP-A59, with a 0.13-kb region derived from MHV-A59, failed to replicate. Interestingly, replication of MRP-A59 was temperature dependent; it occurred at 39.5 degrees C but not at 37 or 35 degrees C, whereas a DI RNA with an MHV-JHM-derived 0.13-kb region replicated at all three temperatures. At 37 degrees C, synthesis of MRP-A59 negative-strand RNA was detected in MHV-infected and MRP-A59 RNA-transfected cells. Another DI RNA with the internal 0.13-kb region deleted also synthesized negative-strand RNA in MHV-infected cells. MRP-A59-transfected cells were shifted from 39.5 to 37 degrees C at 5.5 h postinfection, a time when most MHV negative-strand RNAs have already accumulated; after the shift, MRP-A59 positive-strand RNA synthesis ceased. The minimum sequence required for maintenance of the positive-strand major stem-loop structure and biological function of the MHV-JHM 0.13-kb region was about 57 nucleotides. Function was lost in the 50-nucleotide sequence that formed a positive-strand stem-loop structure identical to that of MHV-A59. These studies suggested that the RNA structure made by the internal sequence was important for positive-strand MHV RNA synthesis.     

Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA.

A protein similar to that previously demonstrated on poliovirus RNA and replicative intermediate RNA (VPg) was found on all sizes of nascent viral RNA molecules and on the polyuridylic acid isolated from negative-strand RNA. 32P-labeled nascent chains were released from their template RNA and fractionated by exclusion chromatography on agarose. Fingerprint analysis using two-dimensional polyacrylamide gels of RNase T1 oligonucleotides derived from nascent chains of different lengths showed that a size fractionation of nascent chains was achieved. VPg was recovered from nascent chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5' terminus could be demonstrated on nascent RNA, and the yield of VPg was consistent with one molecule of the protein on each nascent chain. These results are consistent with the concept that the protein is added to the 5' end of the growing RNA chains at a very early stage, possibly as a primer of RNA synthesis. Analysis of the polyuridylic acid tract isolated from the replicative intermediate and double-stranded RNAs indicated that a protein of the same size as that found on the nascent chains and virion RNA is also linked to the negative-strand RNAs. It is likely that a similar mechanism is responsible for initiation of synthesis of both plus- and minus-strand RNAs.     

A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro

Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.     

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.     

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.     

Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor

The hydrophobic NH2 terminus of F1 (FRED) of the simian virus 5 fusion (F) protein is implicated in mediating cell fusion, but in the inactive F0 precursor the FRED is translocated across membranes. Hybrid proteins containing the FRED as a potential membrane anchorage domain and a mutant of F0 lacking the preceding five-arginine cleavage/activation site were used to study the effect of position on the FRED. The experiments indicate that the SV5 F protein has evolved an exquisite control system for biological activity: the FRED is close to the threshold of hydrophobicity required to function as a membrane anchor. The FRED is not sufficiently hydrophobic to halt translocation when in an internal position, but on cleavage/activation the threshold of hydrophobicity is effectively lowered, and the FRED, now the NH2 terminus of F1, is able to interact stably with membranes.     

Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix.

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.