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

Abstract

Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription 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 analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected 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 fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs (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, seven 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 yielded 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. (iii) Cellular RNAs detected in virions were representative of the abundant cellular RNAs. Last, RNA extracted from virions was translated in vitro and the translation products were reacted with antibody to alphaTIF (VIP16). The immune precipitate contained a labeled protein with the apparent molecular weight of alphaTIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent selectivity in the packaging of the viral RNAs packaged in virions is unknown.

FIG. 1

Primer sequences.

FIG. 2

Photographs of autoradiographic images of HSV-1(F) cDNA array. The HSV-1 cDNA arrays representing the known ORFs were constructed as described in Materials and Methods. A total of 75 cDNA arrays were imprinted in duplicate as described, and the identity of each spot is listed in Table 2. (A and B) The arrays were hybridized with ³²P-labeled cDNAs derived from poly(A)⁺ RNA extracted from infected HEp-2 or rabbit skin cells, respectively. (C) The same cDNA array was hybridized with ³²P-labeled cDNA derived from RNA extracted from virions purified from extracellular medium of infected HEp-2 cells.

FIG. 3

Photographs of DNA bands derived by reverse transcription and PCR amplification of RNAs extracted from HSV-1(F) virions using primers of selected ORFs. RNAs derived from HSV-1(F) virions was reverse transcribed as described in Materials and Methods. The PCR was performed with primers specific for the HSV-1 ORFs indicated. The selected ORFs were those of RNAs detected in all virion preparations. Vero/I and HEp-2/I, virions purified from homogenized infected cells; HEp-2/E, virions purified from extracellular medium. Lanes RT⁺, presence of RT in the RT mixture; lanes RT⁻, control reactions in which RT was omitted; lanes CTR⁺, plasmid DNA containing the sequence of the indicated ORF was used as template; lanes CTR⁻, no template was added to the PCR mixture; lanes MW, 1-kb DNA ladder used as a size marker.

FIG. 4

Photographs of DNA bands derived by reverse transcription and PCR amplification of RNAs extracted from R7023 mutant virions using primers of selected ORFs. The selected ORFs were those of RNAs detected in all virion preparations. Note that the R7023 mutant lacks the genes U_S8 through U_S12. The procedures were the same as those described in the legend to Fig. 3. Lanes RT⁺, presence of RT in the RT mixture; lanes RT⁻, control reactions in which RT was omitted; lanes CTR⁺, plasmid DNA containing the sequence of the indicated ORF was used as template; lanes CTR⁻, no template was added to the PCR mixture; lanes MW, 1-kb DNA ladder used as a size marker.

FIG. 5

Autoradiographic image of a human cDNA array hybridized to labeled cDNAs derived from cellular or virion RNAs. (A) Human cDNA array (GF2111; Research Genetics) membrane was probed with ³³P-labeled cDNA generated by reverse transcription of total RNA isolated from HSV-1-infected HEp-2 cells 22 h after infection. The same membrane was probed with ³²P-labeled cDNA generated by reverse transcription of RNA isolated from intracellular (B) or extracellular (C) purified HSV-I(F) virions from infected HEp-2 cells. Arrows indicate relatively abundant mRNA species consistently positive in all preparations.

FIG. 6

Autoradiographic images of in vitro translation of purified virions RNAs and subsequent immunoprecipitation with UL48 antibody. Lane 1, autoradiographic image of electrophoretically separated products of in vitro translation of virion RNAs; lane 2, electrophoretic mobility of the in vitro translation mixture to which no exogenous RNA was added (negative control); lane 3, autoradiographic image of the electrophoretically separated immunoprecipitate of the translation products shown in lane 1 obtained with the anti-U_L48 antibody; lane 4, the autoradiographic image of electrophoretically separate precipitate obtained from the translation products shown in lane 2 with the anti-U_L48 antibody.