We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 390C relative to that at 33.50C was 5 x 10'. The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that a and ,B polypeptides were produced, whereas most y polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 103 base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a /8 polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells. ucts have been mapped (21, 23). Several /3 genes have been shown to cluster in the region of the genome between 0.30 and 0.42 map units on the 191 on September 29, 2020 by guest http://jvi.asm.org/ Downloaded from 192 CONLEY ET AL. physical map of the HSV-1 genome (23). To delineate the function of some of these genes, we have selected for mutagenesis a DNA fragment mapping between 0.31 and 0.41 map units. MATERIALS AND METHODS Viruses and cells. The isolation and relevant properties of HSV-1(F) (8), HSV-1(imiP) (11), and HSV-2(G) (8) strains were published elsewhere. Virus stocks were prepared and titrated on Vero or HEp-2 cells as previously described (30). Rabbit skin cells were originally obtained from J. McLaren. Human embryonic lung (HEL) cells were originally obtained from Flow Laboratories, Rockville, Md. Enzynmes and radioisotopes. The restriction endonucleases BamHI and KpnI were obtained from

This report concerns the stable viral RNA sequences that accumulate in HEp-2 cells infected with herpes simplex virus type 1. By hybridizing labeled total DNA and restriction endonuclease DNA fragments with excess unlabeled total nuclear and cytoplasmic RNA, we determined the genetic complexity of the RNA and we mapped the regions on the physical map of herpes simplex virus type 1 DNA that are homologous to the RNA. Our results show the following. (i) The viral RNAs accumulating in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of either cycloheximide or emetine were homologous to 33 and 12% of viral DNA, respectively. All of the fragments tested contained sequences homologous to nuclear RNA. However, only the fragments mapping between 0.00 and 0.18, and 0.53 and 1.00 map units contained sequences homologous to cytoplasmic RNA. (ii) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of phosphonoacetic acid were homologous to 39 and 26% of viral DNA, respectively. In this instance all of the fragments except those mapping between 0.42 and 0.53 map units contained sequences homologous to cytoplasmic RNA. (iii) The viral RNAs that accumulate in the nucleus and cytoplasm 8 h after infection were homologous to greater than 50 and 41%, respectively. Al of the fragments tested contained sequences homologous to cytoplasmic RNA. (iv) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of canavanine are homologous to 33 and 19% of viral DNA, respectively. All of the fragments tested contained sequences homologous to both nuclear and cytoplasmic RNAs. Our results indicate the following. First, there are at least three phases of transcription of viral DNA. Phase 1 does not require the synthesis of host cell or viral proteins. Phase 2 requires the synthesis of viral proteins made before the initiation of viral DNA synthesis. Phase 3 appears to be related to the initiation of viral DNA synthesis. Second, both the extent of transcription and the accumulation of viral RNA in the cytoplasm are tightly regulated. The genetic complexity of total RNA accumulating in infected cells increased in each successive phase. Moreover, the genetic complexity of nuclear RNA was invariably higher than that of cytoplasmic RNA in each phase. Lastly, the results of the studies on viral RNA accumulating in canavanine-treated cells reinforce the hypothesis made previously that more than one polypeptide in each of the a and ,8 polypeptide groups is involved in the transcription preceding the transitions from a to / and /8 to y polypeptide synthesis, respectively, and that canavanine selectively inactivated subsets of these polypeptides permitting only partial transitions from a to /8 and /8 to y to occur. This report deals with the transcriptional pro-DNA. The major emphasis of the report is on gram of herpes simplex virus type 1 (human evidence indicat...

Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection.

Fractionation of polyadenylated RNA from cells infected with herpes simplex virus by affinity chromatography on columns of poly (U) immobilized on glass-fiber filters yielded three major classes of RNA-containing poly(A) chains with average lengths of 30, 50, and 155 adenylate residues [poly(A)30, poly(A)50, poly(A)155]. In contrast, nitrocellulose membranes bound predominantly a fraction of RNA containing poly(A)155. The distribution of cytoplasmic RNA in the three classes was found to be independent of the labeling interval, ranging from 10 min to 6 h. Cytoplasmic poly(A) RNA consisted mainly (57 to 68%) of the poly(A)155 class; this was also the major class (68%) of polyadenylated RNA found in polyribosomes. Nuclear poly(A) RNA consisted largely (42 to 50%) of poly(A)30 class, whereas high-molecular-weight nuclear RNA sedimenting at greater than 45S contained almost exclusively the poly(A)30 tracts. Hybridization experiments involving unlabeled RNA and labeled viral DNA demonstrated the presence of viral RNA sequences complementary to approximately 40% of viral DNA in all polyadenylated RNA classes. Inasmuch as unfractionated cytoplasmic RNA arises from approximately 40% of the viral DNA, we conclude that most, if not all, viral RNA species present in the cytoplasm are adenylated. In contrast to these results, only a fraction of poly(A)155 RNA, complementary to 21% of viral DNA, bound to nitrocellulose filters. The selective binding of poly(A)155 sequences to nitrocellulose filters might be related to its secondary structure, since transcripts homologous to 40% of viral DNA bind to nitrocellulose membranes, provided the RNA is denatured prior to filtration. The data suggest that poly(A) tracts arise by at least two separate steps. The first involves the appearance of poly(A)30 tracts in the high-molecular-weight nuclear transcripts. The second involves polyadenylation to ply(A)50 and poly(A)155 RNA classes concomitant with processing and transport to the cytoplasm.