RNAs extracted from herpes simplex virus 1 virions: Apparent selectivity of viral but not cellular RNAs packaged in virions

Following the lead of recent studies on the presence of RNA in virions of h uman cytomegalovirus, we investigated the presence and identity of RNAs fro m purified virions of herpes simple virus 1. To facilitate these studies, w e designed primers for all known open reading frames (ORFs) and also constr ucted cDNA arrays containing probes designed to detect all known transcript s. In the first series of experiments, labeled DNA made by reverse transcri ption of poly(A)(+) RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analys is of the RNA extracted from purified extracellular virions derived from in fected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNA s from purified intracellular or extracellular virions derived from infecte d HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fractio n of viral ORF transcripts were represented in virion RNA, and only nine RN As (U(L)10, U(L)34/U(L)35, U(L)36, U(L)42, U(L)48, U(L)51, U(S)1/U(S)1.5, U (S)8.5, and U(S)10/U(S)11) were positive in all RT PCR assays. Of these, se ven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the U(S)8 to U(S)12 genes yielde d results similar to those described above, indicating that U(S)11, a known RNA binding protein, does not play a role in packaging RNA in virions. (ii i) Cellular RNAs detected in virions were representative of the abundant ce llular RNAs. Last, RNA extracted from virions was translated in vitro and t he translation products were reacted with antibody to alpha TIF (VIP16). Th e immune precipitate contained a labeled protein with the apparent molcular weight of alpha TIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent sel ectivity in the packaging of the viral RNAs packaged in virions is unknown.