Human cytomegalovirus encodes three G protein-coupled receptor homologues.

Human cytomegalovirus (HCMV) is a herpesvirus with a genome of 230 kilobases (Kb) encoding about 200 genes. Although infection is generally innocuous, HCMV causes serious congenital and neonatal disease, and is a dangerous opportunistic pathogen in immune-deficient individuals. We have identified a family of three HCMV genes which encode polypeptides containing seven putative membrane-spanning domains, and a series of well-defined motifs characteristic of the rhodopsin-like G protein-coupled receptors (GCRs). By these criteria all three of the HCMV sequences are homologous to cellular GCRs. Members of this receptor family function in visual signal transduction, regulation of homeostasis, and development, and include known and potential oncogenes. These receptors are activated by photons or small molecules such as neurotransmitters, and glycoprotein hormones. The finding of viral-encoded GCR homologues implies a further level of complexity in the interactions between HCMV and its host, and may provide a potential pathway for virally transformed cell proliferation. Their identification could permit the development of a novel class of antiviral drugs analogous to beta-adrenergic receptor antagonists.

Asymmetry and polarity of nucleosomes in chicken erythrocyte chromatin.

Nucleosome dimers containing, on average, a single molecule of histone H5 have been isolated from chicken erythrocyte nuclei and the associated DNA fragments cloned and sequenced. The average sequence organization of at least one of the two nucleosomes in the dimers is highly asymmetric and suggests that the torsional, as well as the axial, flexibility of DNA is a determinant of nucleosome positioning. On average the nucleosome dimer is a polar structure containing linker DNA of variable lengths. The sequences associated with H5 containing nucleosomes and core particles are sufficiently different to indicate that removal of histone H5 (or H1) from chromatin may result in the migration of the histone octamer and a consequent exposure of sites for regulatory proteins.

An immediate early gene of human cytomegalovirus encodes a potential membrane glycoprotein.

The sequence of a region of the HCMV genome transcribed during the immediate early (IE) transcriptional phase has been determined. Transcription analysis of this region at the junction between fragments HindIII Z and J has identified three moderately abundant mRNAs of 3.4, 1.7, and 1.65 kb. The 3.4-kb RNA is expressed only under IE conditions of infection. It is composed of four exons and its predicted translation product has features characteristic of a membrane-bound glycoprotein. The 1.7-kb RNA is transcribed at both IE and late times postinfection. It is expressed from the same promoter as the 3.4-kb RNA and does not appear to be spliced. The 1.65-kb RNA is present at the IE phase, but is more abundantly transcribed at late times. It is composed of two exons and is 3' coterminal to the 3.4-kb RNA. Within the predicted translation product of the 1.65-kb RNA there are three regions which show homology to a family of related open reading frames found within the short unique region of HCMV.

Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus.

Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.

Large-scale rearrangement of homologous regions in the genomes of HCMV and EBV.

The 20,349-bp sequence of the human cytomegalovirus (HCMV) HindIII F fragment has revealed eight open reading frames with homology to herpes simplex virus (HSV) and/or Epstein-Barr virus (EBV). With respect to EBV, these homologous genes can be divided into two blocks: one block contains three genes, including the DNA polymerase and glycoprotein B, and the other block contains five genes of unknown function. Although the relative organisation of genes within each block is identical in HCMV and EBV, the relative position of each block within the two genomes differs: in HCMV the two blocks are present directly adjacent to each other, whereas in EBV they are found 92 kb apart. This suggests that a genetic rearrangement has occurred in this region. Transcription analysis of the glycoprotein B gene is presented and the evolutionary relationship between the genomes of HCMV, EBV, and HSV is discussed.

Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene.

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. We present here a 5280-base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, we were able to analyze the extent of homology between the DNA polymerases of three distantly related herpesviruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpesviruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type 1 and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

Sequence periodicities in chicken nucleosome core DNA.

The rotational positioning of DNA about the histone octamer appears to be determined by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, we have analysed the sequences of 177 different DNA molecules from chicken erythrocyte core particles. All variations in the sequence content of these molecules, which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier analysis has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addition, it is observed that long runs of homopolymer (dA) X (dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-centre of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA molecule of defined sequence may be strongly influenced by the placement of (dA) X (dT) sequences. It may also be influenced by any aversion of the protein for sequences in the "linker" region, the sequence content of which remains to be determined.

The short unique region of the B95-8 Epstein-Barr virus genome.

The 12-kbp short unique region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for latent and lytic cycle transcripts. Two latent and three late mRNAs have been detected, the largest of the late transcripts potentially encoding a 143-kDa protein. The region containing oriP, the putative origin of replication of the genome as a plasmid in latently infected B lymphocytes, is shown to contain 21 direct repeats of a 30-bp A+T-rich sequence and a related large inverted repeat.

DNA sequence and expression of the B95-8 Epstein-Barr virus genome.

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.

DNA sequence analysis of the EcoRI Dhet fragment of B95-8 Epstein-Barr virus containing the terminal repeat sequences.

An analysis of the approximately 12,440 base-pair sequence of the EcoRI Dhet fragment isolated from the circular episomal form of the B95-8 strain of Epstein-Barr virus is presented. This fragment contains the covalently joined ends of the intracellular episomal form of the molecule. In the viral capsid the DNA is linear and the joining is mediated via the terminal repeated DNA. Four copies of tandem repeated DNA were present in this clone, three with a repeat size of 538 and one of 523. The positions of a number of possible protein coding regions and transcription signals are discussed. In particular a possible spliced coding region for an approximately 45,000 Mr protein expressed in latently infected transformed cells is proposed. The predicted protein sequence contains hydrophobic regions separated by charged amino acids reminiscent of a membrane protein.