Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant

Abstract

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40 degrees C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40 degrees C. In Aedes albopictus cells at 34.5 degrees C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40 degrees C ( approximately 140%) and in Aedes cells at 34.5 degrees C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.

FIG. 1.

Schematic representation of the strategy used to construct hybrid genomes. cDNA copies of the SstI-to-Eco47III region of revertant genomes (hatched boxes) were exchanged for the corresponding fragment in the nsP4 Ser183 (crosshatched boxes) or the Toto1101 (open boxes) genomic backgrounds.

FIG. 2.

RNA accumulation at 40°C by Toto1101 and 1His:4Arg at 40°C. Triplicate CEF cultures were infected with Toto1101 (open boxes) or 1His:4Arg (shaded boxes) at an MOI of 100 at 30°C and were shifted to 40°C at 1 h p.i. Cultures were labeled continuously with 100 μCi of [³H]uridine/ml in the presence of 20 μg of actinomycin D/ml from 1 to 8 h p.i., when the cells were harvested. (A) Total RNA. Aliquots of each total cell lysate were analyzed for acid-insoluble incorporation. (B) RF RNA. RF RNA cores of the viral RIs were isolated as described in Materials and Methods, and their incorporation of radiolabel was determined. (C) Minus-strand RNA. The amount of radiolabeled RF RNA that was in minus strands was determined in nuclease protection assays. (D) Synthesis of plus-strand 49S and 26S RNA relative to RI RNA. The migration positions on agarose gels of RI RNA and 49S and 26S plus-strand RNA were visualized by electrophoresis and autoradiography of total cell lysates as described in Materials and Methods. After the gel areas containing each species were cut and counted, the ratio of the incorporation in 49S plus 26S plus-strand RNA to the incorporation in RI RNA was calculated and is shown.

FIG. 3.

Kinetics of Toto1101 and 1His:4Arg minus-strand syntheses at 40°C. (A) CEF cultures were infected with Toto1101 (•), 1Ile:4Arg (▴), or 1His:4Arg (▪) at an MOI of 100 at 30°C and were shifted to 40°C at 1 h p.i. Cultures were labeled for 1-h pulses from 1 to 7 h p.i. with 200 μCi of [³H]uridine/ml in medium containing 20 μg of actinomycin D/ml. Cells were harvested at the end of the pulse period. Viral RF RNA was isolated, and the percentage of radiolabeled RF RNA in minus strands was determined as described in Materials and Methods. (B) Cycloheximide treatment prevents minus-strand synthesis by nsP1 recombinant viruses. Cultures infected as described for panel A were incubated in the absence of cycloheximide (solid lines) or with 100 μg of cycloheximide/ml (dashed lines) beginning at 2.5 h p.i. and during the labeling period. Incorporation in the minus-strand RNA component of the viral RI and native RF was determined as described in Materials and Methods.

FIG. 4.

Alphavirus nsP1 sequence comparison in the region containing SIN amino acids 335 to 397. The relative positions of the nsP1 suppressors, the causal lesion for ts11 (14), and the suppressor for nsP4 Tyr1→Ala (41) are indicated. Residues identical to the SIN sequence are denoted by asterisks. Sources for sequence data are as follows: SIN and Semliki Forest virus (SFV), ; Whatora virus (WHA), Ellen Strauss, personal communication; Mayora virus (MAY), Ellen Strauss, personal communication; Venezuelan equine encephalitis virus (VEE), ; eastern equine encephalitis virus (EEE), ; Aura virus, ; O'nyong-nyong virus (ONN), ; Ross River virus (RR), ; and Barmah Forest virus (BF), .