Colinearity between the DNAs of Epstein-Barr virus and herpesvirus papio.

Epstein Barr virus (EBV) and herpesvirus papio (HVPapio) DNAs share a common format and 40% homology. Labeled cloned fragments of EBV DNA were hybridized to blots of XbaI, EcoRI, HindIII, and SalI fragments of HVPapio DNA. EBV fragments which mapped from 2 x 10(6) to 54 x 10(6) and from 59 x 10(6) to 106 x 10(6) daltons hybridized to fragments at identical map positions in HVPapio DNA. Regions of nonhomology were demonstrated at 0 x 10(6) to 2 x 10(6), 54 x 10(6) to 59 10(6), and 106 x 10(6) to 115 x 10(6) daltons.     

Herpesvirus papio DNA is similar in organization to Epstein-Barr virus DNA.

EcoRI, HindII, SalI, nd XbaI restriction endonuclease maps of herpesvirus papio (HVPapio) DNA were derived by determining the fragment sizes and the linkage relationships between fragments generated by the different enzymes. The data indicate that HVPapio DNA has a single molecular arrangement which is similar to that of Epstein-Barr virus DNA. The size of the DNA was 110 X 10(6) to 114 X 10(6) daltons. Restriction fragments from both ends varied in the number of repeats of a 4 X 10(5)-dalton sequence, TR, and hybridized to each other. This suggests that there is an identical repeating unit, TR, at both ends of the DNA. There were usually six tandem repetitions (range, 1 to 11) of a 2 X 10(6)-dalton sequence, IR, within the DNA. IR separated the DNA into two domains of largely unique sequence complexity, a 9 X 10(6)-dalton segment, Us, and an 88 X 10(6)-dalton segment, UL. There was homology between DNA fragments which mapped at 25 X 10(6) to 29 X 10(6) to 91 X 10(6) to 95 X 10(6) daltons in UL.     

Epstein-Barr virus DNA. IX. Variation among viral DNAs from producer and nonproducer infected cells.

A comparative analysis of three Epstein-Barr virus DNAs from American patients with infectious mononucleosis (B95-8, Cherry, and Lamont) and four Epstein-Barr virus DNAs from African patients with Burkitt lymphoma (AG876, W91, Raji, and P3HR-1) indicated that the usual format of Epstein-Barr virus DNA includes a variable number of direct repeats of a 0.35 X 10(6)-dalton sequence (TR) at both ends of the DNA, a 9 X 10(6)-dalton sequence of largely unique DNA (Us), a variable number of repeats of a 2 X 10(6)-dalton sequence (IR), and a 89 X 10(6)-dalton sequence of largely unique DNA (UL). Within UL there was homology between DNA at 26 X 10(6) to 28 X 10(6) daltons and DNA at 93 X 10(6) to 95 X 10(6) daltons. The relative sequence order (TR, US, IR, UL, TR) did not vary among "standard" Epstein-Barr virus DNA molecules of each isolate. B95-8 DNA had an unusual deletion extending from 91 X 10(6) to 100 X 10(6) daltons, and P3HR-1 DNA had an unusual deletion extending from 23.5 X 10(6) to 26 X 10(6) daltons. There was sufficient variability among the EcoRI and BamHI fragments of the DNAs to identify each isolate specifically. However, we discerned no distinguishing features for the two geographic or pathogenic origins of the seven isolates. Three intracellular DNAs (Raji, Lamont, and Cherry) and one virion DNA (P3HR-1) were heterogenous in molecular organization and had subpopulations of rearranged or defective molecules. Some regions, particularly 59 X 10(6) to 63 X 10(6) daltons and sequences around TR, frequently participated in rearrangements. Restriction endonuclease maps of the standard and rearranged DNAs of the seven isolates are presented.     

Deletions in the genomes of pseudorabies virus vaccine strains and existence of four isomers of the genomes

As part of a study designed to identify the genes responsible for the virulence of pseudorabies virus, we have mapped the genomes of two independently derived vaccine strains (Bartha and Norden) by restriction enzyme analysis. The structures of these genomes have been compared with that of the genome of a laboratory strain previously mapped, of restriction fragments which had been cloned. The genome of the Bartha strain was found to be very similar to that of other pseudorabies virus strains, except that a deletion of approximately 2.7 X 10(6) daltons was found in the unique short (US) region. This deletion was also observed in the genome of the Norden vaccine strain but was not observed in the genomes of any other pseudorabies virus strains that have been studied (more than 20). The genome of the Norden strain differs from that of other pseudorabies virus strains in several other respects as well. The most important difference is that in contrast to all other pseudorabies virus strains analyzed to date, which contain a type 2 herpesvirus DNA molecule (in which the US region only inverts itself relative to the unique long [UL] region), the genome of the Norden strain is a type 3 molecule in which both the US and the UL regions of the genome invert themselves, giving rise to four isomeric forms of the genome. The ability of the UL region to invert itself is probably related to the fact that a sequence normally present in all other pseudorabies virus strains at the end of the UL only is found also in inverted form at the junction of the UL and the internal inverted repeat in the Norden strain.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.     

Replication of Viral Deoxyribonucleic Acid and Breakdown of Cellular Deoxyribonucleic Acid in Epstein-Barr Virus Infection

Infection of Raji cells with Epstein-Barr virus (EBV) causes suppression of cellular deoxyribonucleic acid (DNA) synthesis and fragmentation of the cellular DNA. About 1,000 copies of EBV DNA of normal size (about 5 x 10(7) daltons in a single strand, as shown in an alkaline gradient) are synthesized per cell.     

Evaluation of DNA homology of Marek's disease virus, herpesvirus of turkeys and Epstein-Barr virus under varied stringent hybridization conditions

Marek's disease virus (MDV) showed only 0.3-0.6% homology in DNA sequence with herpesvirus of turkeys (HVT) at Tm-24.4 degrees C in spite of its antigenic similarity to the latter. Southern blot hybridization under stringent conditions showed that the homology between MDV and HVT is located in the restricted portion of these viral genomes. At Tm-49.6 degrees C, which permits the detection of homology with one base mismatch in three between the MDV and HVT DNAs, sequences with weak homology were found to be distributed over most of these viral genomes. No homology was detected between Epstein-Barr virus and either MDV or HVT DNA.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

The complete genome sequence of herpesvirus papio 2 (Cercopithecine herpesvirus 16) shows evidence of recombination events among various progenitor herpesviruses

We have sequenced the entire genome of herpesvirus papio 2 (HVP-2; Cercopithecine herpesvirus 16) strain X313, a baboon herpesvirus with close homology to other primate alphaherpesviruses, such as SA8, monkey B virus, and herpes simplex virus (HSV) type 1 and type 2. The genome of HVP-2 is 156,487 bp in length, with an overall GC content of 76.5%. The genome organization is identical to that of the other members of the genus Simplexvirus, with a long and a short unique region, each bordered by inverted repeats which end with an "a" sequence. All of the open reading frames detected in this genome were homologous and colinear with those of SA8 and B virus. The HSV gene RL1 (gamma(1)34.5; neurovirulence factor) is not present in HVP-2, as is the case for SA8 and B virus. The HVP-2 genome is 85% homologous to its closest relative, SA8. However, segment-by-segment bootstrap analysis of the genome revealed at least two regions that display closer homology to the corresponding sequences of B virus. The first region comprises the UL41 to UL44 genes, and the second region is located within the UL36 gene. We hypothesize that this localized and defined shift in homology is due to recombination events between an SA8-like progenitor of HVP-2 and a herpesvirus species more closely related to the B virus. Since some of the genes involved in these putative recombination events are determinants of virulence, a comparative analysis of their function may provide insight into the pathogenic mechanism of simplexviruses.     

Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections.

The BamHI K region of Epstein-Barr virus DNA is transcribed in latently infected cells from Burkitt tumors and in growth-transformed B-lymphocytes latently infected with Epstein-Barr virus. We determined the nucleotide sequence of a 1,153-base pair HinfI fragment in BamHI fragment K from the B95-8 Epstein-Barr virus isolate. The fragment contains a remarkable 708-base pair simple sequence repeat array, designated IR3, which is composed of only three nucleotide triplet elements: GGG, GCA, and GGA. The triplets are organized into three repeat units: GCAGGA, GCAGGAGGA, and GGGGCAGGA. Immediately 3' of IR3 are tandem nearly perfect direct repeats of two different 24-base pair sequences. IR3 is conserved at a colinear position in the DNAs of other Epstein-Barr virus isolates, and a homologous sequence maps at the same location in the genome of a genetically related baboon herpesvirus, herpesvirus papio. IR3 is transcribed from left to right in latently infected, growth-transformed IB4 cells. It encodes part of a 2.0-kilobase exon of the 3.7-kilobase cytoplasmic polyadenylated RNA previously detected in IB4 cells (van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1981). IR3 also encodes parts of 2.4- and 1.0-kilobase RNAs in productively infected B95-8 cells.     

Characterization of guinea pig cytomegalovirus DNA.

The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV). GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation. Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells. The cytopathology was characteristic of that seen after infection with GPCMV. Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1%. The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide. GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient. Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons. The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined. Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

Common and specific properties of herpesvirus UL34/UL31 protein family members revealed by protein complementation assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 beta-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus permits replication of terminal repeat-containing plasmids

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus can associate with mitotic chromosomes and promote latent episome maintenance and segregation. Here we report that LANA also mediates the replication of plasmid DNAs bearing viral terminal repeats. The predicted secondary structure of LANA's C terminus reveals striking similarity to the known structure of the DNA-binding domain of Epstein-Barr virus EBNA1, despite the absence of primary sequence homology between these proteins, suggesting conservation of the key mechanistic features of latent gammaherpesvirus DNA replication.