Similar interactions of the poliovirus and rhinovirus 3D polymerases with the 3' untranslated region of rhinovirus 14

Abstract

We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.

FIG. 1

Structures and sequences of 3′ UTRs. (A) Secondary structure of the PV3 3′ UTR. (B) Structure of the HRV14 3′ UTR showing the sequence change at the base of the stem to generate mut4. (C) Conserved sequence at the base of the 3′ stem of human rhinoviruses. (D) The 3′ UTR sequence remaining after removal of the stem-loop to generate HRV14.Δ3′.

FIG. 2

In vitro-transcribed RNA from poliovirus-based cDNAs serially diluted (10⁰ to 10⁻⁵), transfected into Ohio HeLa cells, and overlaid with semisolid agar. pT7FLC.3′HRV14 (A) has a wt HRV14 3′UTR, and pT7FLC.mut4 (B) has a stem mutant 3′ UTR. Other constructs, based on pT7FLC.mut4, include reverse-transcribed and PCR-amplified fragments from recovered virus. pT7FLC.mut4.X/S (C) and pFLC.mut4.H (D) contain the XbaI/SalI and HindIII fragments, respectively. pT7FLC.mut4.3C (E) and pT7FLC.mut4.3D (F) contain the identified 3C and 3D mutations respectively.

FIG. 3

Construction of PV3- and HRV14-based clones. Shaded areas are reverse-transcribed and PCR-amplified fragments from recovered mut4 virus. (A) All constructs are based on PV3 (Leon) with an HRV14 mut4 3′ UTR (FLC.mut4); the restriction endonuclease sites used are indicated, and the constructs generated are shown. (B) HRV14 cDNAs were constructed by using the restriction sites shown.

FIG. 4

(A) Alignment of amino acids 8 to 36 of the PV3 and HRV14 polymerases. The observed nucleotide and amino acid changes are shown for both. (B to D) Tenfold serial dilutions (10⁰ to 10⁻⁵) of RNA generated in vitro from pT7HRV14 (B), pT7HRV14.mut4 (C), and pT7HRV14.mut4.E (D) with substitution of the EcoRV fragment from recovered virus, transfected into Ohio HeLa cells, and overlaid directly with agar.

FIG. 5

Plaque-purified PV3-HRV14 chimeric viruses were used to infect Ohio HeLa cells at a multiplicity of infection of 10 for a one-step growth curve to compare N₁₈Y viruses. Mut4 has the 3′ stem mutant, and the 3D N₁₈Y mutation is present where indicated. ■, FLC.3′HRV14; ▴, FLC.3′HRV14.3D; ⧫, FLC.mut4.3D.

FIG. 6

One-step growth curve to compare wt HRV14 with HRV14.Δ3′ recovered virus following 5 or 13 passages in tissue culture. ■, HRV14.Δ3′, passage 13; ▴, HRV14.Δ3′, passage 5; ⧫, HRV14. TCID₅₀, 50% tissue culture infective dose.