The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D pol , UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD pro . Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D pol . Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D pol in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn 2؉ as a cofactor compared to Mg 2؉ or other divalent cations.Poliovirus (PV) is a small plus-stranded RNA virus belonging to the family Picornaviridae. The members of this virus family are characterized by the presence of a peptide (VPg) covalently linked to the 5Ј end of the viral genome. Replication is a two-step process, beginning with the synthesis of a complementary minus strand (1). That strand, in turn, serves as a template for the production of progeny plus-strand RNAs. Although the basic steps of RNA replication are well established, very little information is available about the details of these processes. The enzyme primarily responsible for RNA synthesis is the viral RNA polymerase 3D pol , which is both primer and template dependent and possesses two important synthetic activities in vitro (12, 37). The first activity, catalyzing the elongation of an oligonucleotide primer on an RNA template, was discovered Ͼ20 years ago and has been thoroughly characterized (4, 12). The second synthetic activity of 3D pol was only recently identified as a reaction in which UMP is linked to the hydroxyl group of a tyrosine in VPg, yielding VPgpU and VPgpUpU (37, 39). These precursors, which can not only be found in PV-infected cells (8) but can also be made in crude replication complexes (49, 52), are believed to be the primers used by 3D pol for both minus-and plus-strand RNA synthesis.The RNA genome of PV (7,525 nucleotides [nt]) contains a long 5Ј nontranslated region (NTR), a single open reading frame, a short 3Ј NTR, and a poly(A) tail (Fig. 1) (20). VPg is attached to the 5Ј-terminal UMP of the RNA by a phosphodiester bond (2, 45). The linkage is between the 5Ј phosphate of UMP and the hydroxyl group of a tyrosine in VPg (2,26,45).Translation of the RNA results in the synthesis of ...

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The Flavivirus genus of the Flaviviridae family encompasses numerous enveloped plus-strand RNA viruses. Dengue virus (DENV), a flavivirus, is the leading cause of serious arthropod-borne disease globally. The genomes of DENV, like the genomes of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5′-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation, and they are not arthropod transmitted. In 2006, E. Harris’ group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5′ and 3′ termini. Here we present evidence that the DENV or ZIKV 5′ untranslated regions (5′-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped monocistronic mRNAs 5′ terminated with the DENV or ZIKV 5′-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells and second, that either 5′-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK cells but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5′ terminated with pppAN… or GpppAN…, can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5′-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses. IMPORTANCE Members of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue virus [DENV], Zika virus [ZIKV], and yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5′ untranslated region (5′-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.

Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5' NTR and coding sequences of the capsid proteins and viral RNA polymerase, 3D(pol). Previous studies have implicated at least two mutations in 3D(pol) of Sabin 1 vaccine strain [PV1(S)], including a Y73H change, as contributing to these phenotypes. We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the terminal protein VPg with 3D(pol) isolated from PV1(S). Wt and two mutant 3D(pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5 degrees C. Our results show that at 39.5 degrees C the Y73H mutation leads to a defect in the synthesis of VPgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT)(15) primer. The double mutant protein had the same activities as Y73H 3D(pol). Using the yeast two-hybrid assay, we detected a reduced interaction between 3D(pol) molecules carrying either the single or double mutations. Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D(pol). A model will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere with VPg uridylylation. Alternative explanations, however, cannot be excluded at the present time.