In recent years, both cellular and viral RNAs have been reported to be packaged in virions of human cytomegalovirus (CMV) (1, 4, 7). The observations are of particular interest for several reasons. Foremost, human CMVs are among the largest viruses infecting cells of higher organisms. In addition, these viruses, members of the Herpesviridae family, incorporate into their virions numerous proteins with multiple functions that effectively assist in the creation of an effective intracellular environment for rapid takeover and redirection of cellular functions to the benefit of the virus. In comparison with other members of Herpesviridae family, the functions encoded in their genomes and the ready-made proteins brought into cells during infection should be more than sufficient to render the infected cell a very pliant client. The presence of the RNAs in virions is therefore an unexpected, novel, intriguing facet of herpesvirus biology.Following the basic premise that viruses do not perform gratuitous functions, we decided to determine whether herpes simplex virus type 1 (HSV-1) virions also contain RNAs and to determine their function. The advantage of HSV-1 is twofold. First, HSV-1 contains fewer open reading frames (ORFs) and the pattern of transcription of the viral genome has been extensively studied. Second, the major functions of HSV-1 gene products are at least in part understood. Hence, if RNAs were packaged in virions, we would have a basis on which to evaluate the significance of the packaged mRNAs.The purification of HSV-1 virions has been extensively studied and characterized (5, 10). In this report, RNAs extracted from either intracellular or extracellular purified virions after RNase digestion were reverse transcribed, amplified by PCR, and subjected to two kinds of analyses. We report that a fraction of the HSV RNAs are represented in RNAs extracted from purified preparations and that the RNA transcripts of nine ORFs were detected in all purified preparations tested. We also report the presence of cellular RNAs in purified virions. In this instance, the selectivity of the packaged RNAs is less well apparent. MATERIALS AND METHODS Cells and viruses.Vero and HEp-2 cell lines (American Type Culture Collection) and a rabbit skin cell line (originally obtained from J. McLaren) were propagated in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum. HSV-1(F) is the prototype HSV-1 strain used in this laboratory (2). Isolation of the mutant virus R7023 was described elsewhere (6). R7023 lacks the genes U S 8 through U S 12. Titers of the stocks of HSV-1(F) and R7023 on Vero cells were determined.Purification of virions. HSV-1 and R7023 virions were purified as described by Spear and Roizman (10). Briefly, Vero or HEp-2 cells grown in roller bottles or in 150-cm 2 flasks were exposed to 5 PFU of virus per cell. The cells were harvested 22 to 24 h after infection and centrifuged at 2,000 rpm for 10 min in an Allegra 6R centrifuge equipped with a GH-3.8 rotor (Beckman Coulter, Inc., Fuller...

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP

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Herpes simplex virus (HSV)-2 caused a genital ulcer in a 40-year-old allogenic stem cell recipient, and a secondary herpetic whitlow appeared during 2 months of acyclovir (ACV) therapy. Both genital ulcer, and whitlow were cured 3 months later, but 6 months after recovery the whitlow alone recurred. DNA of the genital, first, and recurrent whitlow isolates showed similar endonuclease digestion fragment profiles. The genital virus was ACV-sensitive, and the two whitlow isolates were ACV-resistant/thymidine kinase (TK)-deficient. The TK gene of the whitlow isolates had the same frame shift from the 274th amino acid and termination at the 347th amino acid due to the deletion of a cytosine at the 819th nucleotide. Because the temperature of the thumb is 33/34 degrees C or lower, the temperature sensitivity of the isolates were compared, and both whitlow isolates were significantly more temperature-sensitive (ts) at 39 degrees C than the genital isolate. The two whitlow isolates showed cutaneous pathogenicity in mouse ear pinna but not midflank, while the genital isolate was pathogenic at both sites, suggesting that temperature adaptation was an important element of pathogenicity in the whitlow. The virus populations of isolates of the genital, and first whitlow were examined by 31, and 82 clones, respectively, and the clones from genital, and whitlow isolates were ACV-sensitive, and -resistant, respectively, showing their homogeneity. The acyclovir-sensitive genital lesion had spread as a TK-deficient/ts herpetic whitlow during ACV treatment, and an apparently TK-deficient virus adapted to the local temperature might have caused the whitlow recurrence.

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