Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome

During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.     

Identification of a herpesvirus Saimiri cis-acting DNA fragment that permits stable replication of episomes in transformed T cells.

Herpesvirus saimiri is a lymphotropic herpesvirus capable of immortalizing and transforming T cells both in vitro and in vivo. Immortalized and transformed T cells harbor several copies of the viral genome as a persisting genome. The mapping of the cis-acting genetic cis-acting segment (oriP) required for viral episomal maintenance is reported here. Viral DNA fragments that potentially contain oriP were cloned into a plasmid that contains the hygromycin resistance gene. After several round of subcloning followed by transfection, oriP was mapped to a 1.955-kb viral segment. This viral fragment permits stable plasmid replication without deletion or rearrangement as well as episomal maintenance without integration or recombination. The function of oriP depends on a trans-acting factor(s) encoded by the viral genome. The 1.955-kb viral segment includes a dyad symmetry region located between two small nuclear RNA genes and is located upstream of the dihydrofolate reductase gene homolog. Therefore, this oriP contains novel elements distinct from those of other DNA viruses.     

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.     

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping.

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.     

Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome contains a duplication of a long unique-region fragment within the terminal repeat region

Use of the Kaposi's sarcoma-associated herpesvirus (KSHV) bacterial artificial chromosome 36 (KSHV-BAC36) genome permits reverse genetics approaches to study KSHV biology. While sequencing the complete KSHV-BAC36 genome, we noted a duplication of a 9-kb fragment of the long unique region in the terminal repeat region. This duplication covers a part of open reading frame (ORF) 19, the complete ORFs 18, 17, 16, K7, K6, and K5, and the putative ORF in the left origin of lytic replication, and it contains the BAC cassette. This observation needs to be kept in mind if viral genes located within the duplicated region are to be mutated in KSHV-BAC36.     

Construction of a cloned library of the EcoRI fragments from the human cytomegalovirus genome (strain AD169).

The DNA genome of human cytomegalovirus (HCMV) strain AD169 is 158 x 10(6) Mr. Cleavage of the HCMV DNA with the restriction endonuclease EcoRI yields 35 major fragments ranging in size from 0.54 x 10(6) Mr. We have constructed a cloned library of the EcoRI fragments of this strain of HCMV, using the plasmid pACYC184 and the recipient bacterium Escherichia coli strain HB101 RecA-. The viral origin of the cloned inserts was determined by hybridization to viral DNA. The fragments were characterized further by digestion with other restriction enzymes. Several clones were obtained which contained sequences spanning the junction between the long (L) and short (S) components of the viral DNA sequences. These clones differed in molecular weight by multiples of 0.3 x 10(6) to 0.4 x 10(6) Mr. The variability found in the clones was also reflected in the genome. Each clone containing a junction sequence hybridized to a series of bands on Southern filters of EcoRI-digested HCMV DNA. This "ladder effect" provided evidence for a region of heterogeneity within the L-S junction.     

Spiroplasma citri Virus SpV1: Characterization of Viral Sequences Present in the Spiroplasmal Host Chromosome

Spiroplasma citri virus SpV1-R8A2B is a naked, rod-shaped virus with a circular, single-stranded DNA genome of 8273 bp. SpV1-related sequences were detected in the chromosomal DNA of all S. citri strains tested. Southern blot hybridization analyses revealed that several copies of most, if not all, SpV1 ORFs are present in the chromosome of S. citri strain R8A2. For further study of the integrated viral sequences, a genomic DNA library of S. citri R8A2 was constructed, and two cloned chromosomal DNA fragments containing SpV1 viral sequences were studied by comparison with the free viral genome of SpV1-R8A2B. One fragment seems to contain a full-length viral genome, while the other contains only parts of the viral genome. In both fragments, the left and right ends of viral sequences consist of 31-bp inverted repeat sequences, those which are facing each other at nucleotide 4737 in the circular viral genome. In addition, both fragments contain the SpV1-ORF3, encoding a putative transposase, immediately upstream of the right repeat. These data suggest that the SpV1-ORF3 and the repeat sequences could be parts of an IS-like element of chromosomal origin.     

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.     

Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments

Restriction enzyme XbaI DNA fragments that represent 99% of the sequences from the long and short unique as well as the repeat sequences of the human cytomegalovirus (CMV) genome have been cloned into bacterial plasmid pACYC184. The viral DNA sequences associated with the recombinant plasmids were analyzed by restriction mapping and by hybridization to fragments of authentic viral DNA. The relationship of the cloned viral DNA fragments to the XbaI physical map of the viral genome is demonstrated. Even though large recombinant plasmids ranging from approx. 39 to 1.8 kb were isolated, most if not all of the viral DNA fragments were stable during propagation in Escherichia coli HB101.     

Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences

The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSU) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than 99.48% homologous, and the consensus sequences of 3 different medicinal mushrooms were more than 97.80% homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Identification of the Rhesus Macaque Rhadinovirus Lytic Origin of DNA Replication

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.     

The sequence of the 3.3-kilobase repetitive element fromDipodomys ordii suggests a mechanism for its amplification and interspersion

Summary DNA from the kangaroo rat,Dipodomys ordii, contains a 3.3-kb, highly repeated sequence that is interspersed throughout the genome in small tandem clusters. One 3.3-kb unit has been cloned into pBR322 and the nucleotide sequence determined. The clone used was shown to be representative of the bulk of such sequences found in the genomic DNA. The sequence contains 10 homologous subunits each ca. 260 bp in length. Comparison of these to one another yielded a 258-bp consensus sequence containing a 35-bp terminal inverted repeat. Two unique stretches also occur. One of these contains a region that could serve as a promoter for RNA polymerase III; the other contains a sequence related to the ARS sequences of yeast. It is proposed that an ancestral sequence similar to the consensus sequence was amplified to 10 or more units, and that, subsequently, two other sequences were inserted. The properties of these insertions may have led to the dispersal of the sequence throughout the genome.     

Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.     

Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA.

A 535 base pair DNA fragment which maps entirely within the IRS/TRS regions of the herpes simplex virus type 1 (HSV-1) genome and contains all the cis-acting signals necessary for it to function as an origin of viral DNA replication has previously been identified (N.D. Stow and E.C. McMonagle, Virology, in press). When BHK cells were transfected with circular plasmid molecules containing cloned copies of this DNA fragment, and superinfected with wt HSV-1 as helper, amplification of the input plasmid was detected. Two observations indicated that the amplified DNA was not packaged into virus particles. Firstly, when the transfected cells were disrupted the amplified DNA was susceptible to digestion by added DNase, and secondly, it was not possible to further propagate the DNA when virus from the cells was passaged. Fragments from the joint region and from both termini of the viral genome were inserted into origin-containing plasmids and the resulting constructs analysed. In all cases the inserted fragment allowed the amplified DNA to be further passaged, and a proportion to become resistant to digestion with DNase. These observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.