Table 1.

Replicon and virus phenotypes of nsP2-726 mutants

MutationaRNA level (%)bProcessing (mean ± SEM)cCPE
ReplicondViruse
726Pro (wild type)1001YesYes
726Ala1010.77 ± 0.13YesYes
726Val300.32 ± 0.06YesYes
726Gly170.28 ± 0.06YesNof
726Ser9.90.16 ± 0.04YesNof
726Thr5.10.12 ± 0.03YesNof
726Phe1.60.09 ± 0.01Nog
726Tyr1.40.09 ± 0.01No
726Leu1.10.10 ± 0.01No
726Arg1.20.10 ± 0.03No
726Gln0.80.08 ± 0.03No
779LysNDh 0.10 ± 0.01NoND
614AspND0.20 ± 0.03NDLethal
  • a Amino acid substitutions at nsP2 residue 726, residue 779 (S24), or residue 614 (48).

  • b Genomic RNA accumulation for replicon derivatives relative to that for the parent as determined from the data presented in Fig. 7A. Bands were localized by fluorography and excised, and the level of incorporation was determined by liquid scintillation counting.

  • c Results of processing-phenotype assays by cell-free translation, compared by analyzing the ratio of uncleaved P123 to nsP2 (Fig. 7D).

  • d BHK cells were transfected with the various replicons, puromycin was added after 5 h, and the remaining cells were stained with crystal violet after 48 h. Replicons with Phe, Tyr, Leu, Arg, or Gln at position 726 were noncytopathic and produced healthy monolayers in the presence of puromycin. For the other replicons, including the parent (726Pro), the vast majority of the cells were dead by 48 h. As shown in Fig. 7C, at various frequencies, Purr foci were observed for these replicons.

  • e Virus-induced CPE was observed after transfection with Toto1101 derivatives containing the nsP2-726 substitutions. By 24 h posttransfection, complete CPE was observed for the Toto1101 parent and the Ala and Val substitution mutants. The nsP2 hyperprocessing mutation at residue 614 is lethal for virus replication (48).

  • f Transient CPE, recovery, and persistently infected cultures were produced after transfection with RNAs containing Ser, Gly, or Thr.

  • g —, for the Phe, Tyr, Leu, Arg, and Gln mutants, the viral cytopathic phenotype was difficult to assay due to the rapid appearance of cytopathic revertants or pseudorevertants.

  • h ND, not determined.