Characterization of early gene transcripts of molluscum contagiosum virus

Molluscum contagiosum virus (MCV), a member of the family Poxviridae, repli cates well in vivo but cannot be propagated in cell culture. The coding cap acity of the MCV genome was previously determined by DNA nucleotide sequenc e analysis. The objective of the present study was to establish experimenta l systems for the identification and characterization of early MCV gene tra nscripts. MCV mRNA was obtained in three ways: (1) MCV early mRNA was synth esized in vitro using permeabilized virions, (2) MCV mRNA was extracted fro m MCV-infected skin tissue, and (3) MCV mRNA was extracted from MCV-infecte d human embryonic fibroblasts. RNA/DNA hybridization experiments showed sig nificant early transcriptional activity in two parts of the MCV genome. Tra nscripts of 11 early MCV genes located in these parts of the genome, includ ing two subunits of the MCV DNA-dependent RNA polymerase (mc077R and mc079R ), the MCV poly(A)(+) polymerase gene (mc076R), and the MCV MHC class I hom olog (mc080R), were detected in reverse transcription-polymerase chain reac tion experiments. Total RNA obtained from MCV-infected skin tissue was used to confirm these results. Three MCV early transcripts, mc002L, mc004.1L, a nd mc005L, produced distinct bands on rapid amplification of their 3' ends (3' RACE). The 5' mapping of transcription start sites of MCV open reading frames (ORFs) mc002L, mc004.1L, mc005L, and mc148R revealed that the MCV RN A polymerase transcription start sites are consistently located between 11 and 13 nucleotides downstream of the early MCV consensus promoter signal. W hen cDNA from both 5' and 3' mapping experiments was analyzed, MCV ORFs mc0 04.1L and mc005L were found to be transcribed as a single bicistronic mRNA. The transcript from MCV ORF mc066L, encoding a glutathione peroxidase, was detected in in vitro synthesized MCV mRNA as well as in total RNA from MCV -infected human embryonic fibroblasts and MCV-infected skin. This indicates that despite the tack of an early MCV consensus promoter signal immediatel y proximal to the start codon, this particular gene is transcribed early du ring MCV infection. (C) 1999 Academic Press.