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.

Although the m7G5'ppp5'N(m)pNp "capping" group has been found on virtually all known mammalian mRNAs (1), poliovirus polyribosomal RNA has pUp at its 5'-end (2-4). Because poliovirion RNA will direct viral protein synthesis in a cell-free system (5), it was thought to be identical to polyribosomal poliovirus RNA. Instead, we found that less than 10% of the virion molecules contained a pUp 5'-end (2).Lee et al. (6) recently presented evidence that a protein might be linked to the 5'-terminus of poliovirion RNA. We also have obtained evidence for a 5'-terminal protein in virion RNA. When total RNase digests of 32P-labeled virion RNA were examined by paper ionophoresis at pH 3.5, some labeled material was found moving toward the cathode, the direction opposite that of pure nucleotides. This material had the properties of a protein-pUp 5'-terminus of the RNA, a structure also suggested by Lee et al. (6). We have further found that cellulose acetate electrophoresis of a RNase T1 digest of virion RNA separates a protein-linked oligonucleotide. We show here that it consists of the structure protein-pU-U-A-A-A-A-C-A-G, which appears to be the 5'-terminus of poliovirion RNA

The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive-but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3 poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.Poliovirus is a prototypic positive-strand RNA virus with a single-stranded genome that contains a 3Ј-terminal poly(A) tail and a 5Ј-terminal covalently linked protein, VPg (1,15,27). Once released into the cytoplasm of infected cells, poliovirion RNA serves as an mRNA for translation and then as a template for negative-strand synthesis. Multiple rounds of positivestrand synthesis on each molecule of negative-strand RNA result in the synthesis of excess genomic RNA. Overall, the ratio of positive-to negative-strand synthesis is greater than 30:1 (10, 24), which is essential for the production of high yields of virion RNA and progeny virus in infected cells.cis-active replication sequences that are present at the 3Ј and 5Ј ends of poliovirion RNA play an important role in regulating poliovirus RNA replication. The 3Ј nontranslated region (NTR) and the associated poly(A) tail are important cis-active determinants that are required for efficient negative-strand RNA synthesis (14,21,22,28,30,36). The 5Ј-terminal cloverleaf structure in poliovirus RNA is a multifunctional element that is required in cis for negative-strand synthesis, RNA stability, and VPg uridylylation in membrane replication complexes (8,14,17,23,34). A new cis-acting replication element (cre) was recently identified in the genomes of several picornaviruses (9,11,16,18,20). The cre was originally identified in the P1 capsid coding region of HRV14 [cre(VP1)] (19). Mutational analysis of the cr...

A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy pOly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCI. The solubilized polymerase required both poly(A) and ofigo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A) * oligo(U)dependent DlyU) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U).dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase. An RNA-dependent RNA polymerase (replicase) is found in the cytoplasm of cells infected with poliovirus (1) as well as with other picornaviruses (2, 3) and is apparently responsible for the replication of the viral RNA genome. Previous attempts to purify the poliovirus replicase as a soluble enzyme were limited by the lack of an assay for enzymatic activity with an exogenously supplied RNA template. Studies have been restricted to the purification and characterization of the replicase complexed to the endogenous RNA template (4-7) which was found to be associated with cellular membranes (4,(8)(9)(10). A soluble replicase-template complex could be prepared by detergent treatment of the membrane fraction, followed by precipitation of the complex with 2 M LiCl (4-6). Several host and viral polypeptides were found in the precipitate, the predominant one being noncapsid viral protein 4. Previous attempts to stimulate the replicase activity by the addition of exogenous RNA were not successful (4). A template-dependent replicase has been isolated from encephalomyocarditis virus-infected cells, but it was highly unstable (11).The detailed mechanisms involved in the replication of the single-stranded RNA genome of poliovirus are not known. The synthesis of poliovirus messenger and virion RNA (plus strands) takes place in a structure known as the replicative intermediate (12)(13)(14). The replicative intermediate consists of at least one complete strand of complementary (negative strand) RNA and approximately six nascent chains of plus strand RNA (14). The first step in the formation of the replicative intermediate should be the synthesis of a complete negative strand RNA molecule. During synthesis of the negative strand, assuming that it occurs 5' to 3', the initial event would be copying of the poly(A) at the 3' end of the virion RNA to form the poly(U) found at the 5' end of negative strand RNA (15, 16). Thus, negative strand synthesis should be initiated by a poly(A)-dependent poly(U) polymerase. This reasoning led us to investigate if poliovirus-infected cells ...

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2-T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.