During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.

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The most abundant species of human cytomegalovirus (Towne) immediate early polysome-associated RNA originates from a region of ca. 2.8 kilobases (0.739 to 0.755 map units) within the XbaI-E DNA fragment. These sequences code for a 1.95-kilobase mRNA and are referred to as immediate early coding region one (M. F.

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 Å resolution. The structural similarity of this polymerase to other ␣ polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.

The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 3' ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription.

The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.Human cytomegalovirus (CMV), a member of the' herpesvirus classification group, has a large double-stranded DNA genome of 240 kilobases (kb). The viral genome consists of a long-and short unique region flanked by differept repeat sequences 'that are inverted relative to each other. Four genome arrangements, resulting from the possible combination of inversions of the two sections of the genome, are present in DNA preparations in approximately equal amounts (1-7).At immediate early (IE) times after infection-i.e., in the absence of de novo viral protein synthesis, 88% or more of the viral RNA originates from a region in the long unique component of the viral genome (6,8,9) In Vitro Transcription and RNA Fractionation. In vitro transcription was as described by Manley et al. (14). Recombinant plasmids cut with various restriction enzymes to generate linear templates were at a concentration of 100 ig per ml. Some reactions contained a-amanitin (1 pg/ml; Sigma) to inhibit RNA polymerase II activity. The 32P-labeled RNA was subjected to electrophoresis in 1.5% agarose gels containing 10 mM methylmercury (II) hydroxide as described by Bailey and Davidson (15). Molecular weight standards were 23S (3.3 kb) and 16S (1.7 kb) Escherichia coli rRNA (16), 28S (5.3 kb) and 18S (2.0 kb) human' cell rRNA (17), and approximately 0.160 kb tRNA. To visualize the RNA, the slab gels were stained in a solution containing 0.5 M ammonium acetate, 0.005 M 2-mercaptoethanol, and 1 ,ug of ethidium bromide per ml. The gels were dried and exposed to Kodak XOmat AR film. RNA sizes were interpolated from a standard 659The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.- §1734 solely to indicate this fact.

The capsid of herpes simplex virus type 1 (HSV-1) is composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, which are the products of six HSV-1 genes. Recombinant baculoviruses were used to express the six capsid genes (UL18, UL19, UL26, UL26.5, UL35, and UL38) in insect cells. All constructs expressed the appropriate-size HSV proteins, and insect cells infected with a mixture of the six recombinant baculoviruses contained large numbers of HSV-like capsids. Capsids were purified by sucrose gradient centrifugation, and electron microscopy showed that the capsids made in Sf9 cells had the same size and appearance as authentic HSV B capsids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the protein composition of these capsids was nearly identical to that of B capsids isolated from HSV-infected Vero cells. Electron microscopy of thin sections clearly demonstrated that the capsids made in insect cells contained the inner electron-translucent core associated with HSV B capsids. In infections in which single capsid genes were left out, it was found that the UL18 (VP23), UL19 (VP5), UL38 (VP19C), and either the UL26 (VP21 and VP24) or the UL26.5 (VP22a) genes were required for assembly of 100-nm capsids. VP22a was shown to form the inner core of the B capsid, since in infections in which the UL26.5 gene was omitted the 100-nm capsids that formed lacked the inner core. The UL35 (VP26) gene was not required for assembly of 100-nm capsids, although assembly of B capsids was more efficient when it was present. These and other observations indicate that (i) the products of the UL18, UL19, UL35, and UL38 genes self-assemble into structures that form the outer surface (icosahedral shell) of the capsid, (ii) the products of the UL26 and/or UL26.5 genes are required (as scaffolds) for assembly of 100-nm capsids, and (iii) the interaction of the outer surface of the capsid with the scaffolding proteins requires the product of the UL18 gene (VP23).