| A human cytomegalovirus (HCMV) mutant containing a temperature-sensitive (ts) allele of the immediate-early 2 (IE2) protein has been constructed (BTN tsUL122). The ts allele contains a single substitution mutation, converting the cysteine at amino acid position 510 in the IE2 protein to glycine. In transcription activation assays the IE2-C510G protein is able to activate an SV40 early promoter and the HCMV UL112 promoter at 32.5°C but fails to transactivate at 39.5°C. The BTNtsUL122 virus grows to wild-type titers and replicates viral DNA to wild-type levels at 32.5°C. However, at 39.5°C, the mutant is severely growth restricted and fails to produce any detectable virus. Further examination of the BTN tsUL122 virus at 39.5°C indicates that the virus fails to accumulate viral DNA, yet the mutant virus produces immediate-early (IE) transcripts and protein up to 96h post infection. Analysis of early (E) and late (L) transcripts indicates that the BTNtsUL122 virus fails to produce transcripts for the E genes, UL44 and UL54, and the L gene UL82. We believe that the phenotype of the mutant at the non-permissive temperature is due to the inability of the IE2 protein to activate E promoters required for progression into the E phase of the lytic infectious cycle. This is the first report that IE2 is essential for the switch from the IE to E phase in the context of an infection.; Another HCMV mutant (BTNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the IE2 protein. This mutant grew as well as wild-type at both low and high multiplicities of infection. The mutant activated the major-immediate early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the IE2P mutant virus transactivated an endogenous SV40 early promoter four hours earlier, and to higher levels, than the wild-type virus in infected human fibroblasts. The SUMOylated state of the hypophosphorylated IE2 protein was also examined. |
