Identification of a cis -Acting Replication Element within the Poliovirus Coding Region

Abstract

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 Poliovirus, the archetypal picornavirus, is arguably one of the best characterized of all viruses. The availability of infectious molecular clones (33) has enabled the application of reverse genetics to understand the function of the nonstructural proteins and the noncoding regions (NCR) of the virus genome (reviewed in reference 41). The 7.4-kb single-stranded positive (messenger)-sense RNA genome encodes a single polyprotein and contains the necessary signals for virus replication in the cell cytoplasm. Posttranslational cleavage of the polyprotein by virus-encoded proteases (2A pro and 3C pro ) yields the four capsid proteins (VP1 to VP4), the RNA-dependent RNA polymerase (3D pol ), and the accessory proteins required for replication. The input RNA acts as a template for the synthesis of a negative strand which, in turn, is used as a template for synthesis of genome sense RNA. The virus-encoded protein VPg (3A) is implicated as a protein primer for both positive-and negative-sense strand initiation, as are RNA sequences occupying the 5Ј NCR of the virus genome. A cloverleaf (CL) structure of 88 nucleotides (nt) at the 5Ј end of the genome interacts with the virus 3C pro D pol and either the VPg-containing precursor 3AB or the cellular poly-C binding protein type 2 (PCBP2) (2,3,11,28). This 5Ј NCR ribonucleoprotein complex is required for replication and may also be involved in the suppression of virus translation (10).RNA sequences and structures within the 3Ј NCR presumed to be involved in replication are much less well defined. Chimeric polioviruses in which the 3Ј NCR is replaced by the analogous region of other picornaviruses generally retain viability (35), even though there is little sequence or structural homology between substituted 3Ј NCR....

Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages

“…Interestingly, nt 4473 was the same as in wild-type PV2-Lansing and PV2-W2 as well as PV1 strains Sabin and Mahoney. Protein 2C has two functions in viral RNA synthesis: (i) as an ATPase and (ii) directing replication complexes to cell membranes forming replication complexes (Goodfellow et al, 2000). It is possible that this change partly contributes to the faster replication.…”

A “Slide-back” Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus

“…ATPase (16). This RNA element is similar in size and shape to rhinoviral cre elements such as HRV2 cre(2A) (17) and HRV14 cre(VP1) (18).…”

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

“…This describes the frequency of synonymous codon pairs that does not fit the use of individual codons (16)(17)(18) (1). The loci of three replication-essential hairpin structures, Cre (81), and α and β (23,24), as well as the conserved but not-essential RNase L ci element (25,26), are shown above the PP. The SnaB I and Bgl II restriction sites, used for subcloning the P2 and P3 domains, are indicated.…”