The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative-and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU OH , the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative-and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU OH , however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU OH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU OH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU OH in the asymmetric replication of poliovirus RNA.Poliovirus possesses a single-stranded positive-polarity RNA genome that is 7,441 nucleotides (nt) in length (39). VPg, a virally encoded 22-amino-acid protein, is covalently linked to the 5Ј uridine of poliovirus RNA. When released into the cytoplasm of susceptible cells, VPg is removed from the 5Ј terminus of poliovirus RNA by a host enzyme (1, 2). Following the removal of VPg, viral RNA functions sequentially as an mRNA for viral protein synthesis and then as a template for negative-strand RNA synthesis during viral RNA replication. Translation of the long open reading frame in viral mRNA yields a single polyprotein which undergoes both co-and posttranslational processing, culminating in the synthesis of viral capsid and replication proteins (21,24,32). The viral replication proteins interact with both the viral mRNA and components of the host cell to produce membrane-associated ribonucleoprotein complexes (9-11, 44, 45). cis-active RNA structures found within the 5Ј and 3Ј nontranslated regions (NTRs) of poliovirus RNA mediate the sequential translation and replication of viral RNA (4,8,20,28,38,41). Poliovirus mRNA, like other eukaryotic mRNAs (43), may circularize within messenger ribonucleoprotein complexes. Likewise, because viral negative-strand RNA synthesis requires RNA structures from both the 5Ј and 3Ј NTRs, the template for viral negative-strand RNA synthesis may exist in a circularized conformation via 20). Amplification of viral RNA within membranous replication complexes occurs asymmetrically. Viral RNA is first copi...

Chimeric poliovirus RNAs, possessing the 5 nontranslated region (NTR) of hepatitis C virus in place of the 5 NTR of poliovirus, were used to examine the role of the poliovirus 5 NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3 NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5-terminal cloverleaf and 3 NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.The single-stranded positive-sense RNA genome of poliovirus functions sequentially as an mRNA for viral protein synthesis and then as a template for viral negative-strand RNA synthesis (33). Cytoplasmic extracts from HeLa cells support the sequential translation and replication of poliovirus RNA (5, 31). In the presence of 2 mM guanidine HCl, cell-free translation-replication reactions support the translation of viral RNA and the accumulation of viral preinitiation RNA replication complexes (4, 6). Guanidine HCl reversibly blocks the initiation of negative-strand RNA synthesis by interfering with the function of viral protein 2CATPase (3,38,40). When preinitiation RNA replication complexes are resuspended in reaction mixtures in the absence of guanidine HCl, the preinitiation RNA replication complexes synchronously initiate the synthesis of viral negative-strand RNA (6). Ribosomes translating the viral mRNA within preinitiation RNA replication complexes prevent the synthesis of negative-strand RNA (8,15). Thus, the conversion of viral ribonucleoprotein complexes into preinitiation RNA replication complexes is an important temporally regulated event in the replication of poliovirus RNA.Contemporary models of eukaryotic mRNA translation suggest that the 5Ј and 3Ј nontranslated regions (NTRs) of mRNAs communicate via RNA binding proteins bound to both termini of the mRNA (14,19,42). In particular, poly(A) binding protein bound to 3Ј-terminal poly(A) interacts with eukaryotic initiation factors eIF4G I and II anchored to the 5Ј NTR of mRNA, bringing the 5Ј and 3Ј NTRs into proximity (49). During the course of a poliovirus infection, viral protein 2APro mediates the cleavage of eIF4G I and II and poly(A) binding proteins (11,18,23,24). This leads to the shutoff of cap-dependent host protein synthesis and precludes the ability of eIF4G I and II and poly(A) binding protein to form proteinprotein bridges between the 5Ј and 3Ј termini of mRNAs, including poliovirus RNA. Therefo...

The mammalian Orthoreovirus (mORV) core particle is an icosahedral multienzyme complex for viral mRNA synthesis and provides a delimited system for mechanistic studies of that process. Previous genetic results have identified the mORV 2 protein as a determinant of viral strain differences in the transcriptase and nucleoside triphosphatase activities of cores. New results in this report provided biochemical and genetic evidence that purified 2 is itself a divalent cation-dependent nucleoside triphosphatase that can remove the 5 ␥-phosphate from RNA as well. Alanine substitutions in a putative nucleotide binding region of 2 abrogated both functions but did not affect the purification profile of the protein or its known associations with microtubules and mORV NS protein in vivo. In vitro microtubule binding by purified 2 was also demonstrated and not affected by the mutations. Purified 2 was further demonstrated to interact in vitro with the mORV RNAdependent RNA polymerase, 3, and the presence of 3 mildly stimulated the triphosphatase activities of 2. These findings confirm that 2 is an enzymatic component of the mORV core and may contribute several possible functions to viral mRNA synthesis.

The 59-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 59-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 59 cloverleaf RNA and rendered poliovirus mRNA unstable. A 59-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 59-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 59 cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 59 cloverleaf RNA.

Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein microNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, microNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for microNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (lambda1, lambda2, lambda3, micro2, and sigma2) and nonstructural protein sigmaNS, can associate with microNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in microNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log(10) values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement microNS expression and to rescue infectious yields by ~100-fold. We next used microNS mutants specifically defective at forming factory-matrix structures to show that this function of microNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of microNS prevented rescue of viral growth while causing microNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of microNS, designed to represent microNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of microNS known to be involved in both micro2 and sigmaNS association. Thus, factory-matrix formation is an important, though not a sufficient function of microNS during MRV infection; microNS is multifunctional in the course of viral growth.