Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus

The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity.     

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.     

The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.     

Multiple transforming regions of human cytomegalovirus DNA.

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.     

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.     

Complete sequence and genomic analysis of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.     

人类巨细胞病毒UL133基因在临床低传代株中的多态性研究

人类巨细胞病毒在多次传代后,会表现出不同的毒力水平.与临床低传代株Toledo相比,实验室高传代株AD169缺失了19个开放阅读框(ORF).这19个基因被认为是与HCMV致病性最可能相关的一组基因,研究这些基因的多态性对揭示HCMV致病性的遗传基础具有指导意义.UL133基因是这19个ORF中的一个.以临床低传代株Toledo和Merlin为对照,分析了23个临床病毒株UL133基因的遗传多态性.序列分析表明,UL133基因具有一定的多态性,Toledo株、Merlin株与我们分离到的临床株一起可分为3个基因型:G1、G2和G3.G2、G3型毒株均能导致先天性感染.没有发现UL133基因型与患儿临床疾病的必然关联.     

Transformation by Epstein-Barr virus requires DNA sequences in the region of BamHI fragments Y and H.

Eight independent recombinant Epstein-Barr virus genomes, each of which was a transforming strain, were made by superinfecting cell lines containing Epstein-Barr virus DNA (Raji or B95-8 strain) with a nontransforming virus (P3HR1 strain). A knowledge of the constitution of each transforming recombinant allowed the localization of the defect in the genome of the nontransforming parent to a 12-megadalton sequence within the EcoRI A fragment. Within this region, the nontransforming virus has a deletion of the BamHI Y fragment and about half of the sequences in the adjacent BamHI H fragment. The present data suggest that this deletion is responsible for the nontransforming phenotype. Furthermore, mapping a deletion in one of the recombinant genomes allowed the conclusion that a sequence (comprising about 20% of the Epstein-Barr virus genome) from the center of BamHI-D to BamHI-I' is not necessary for the maintenance of transformation by Epstein-Barr virus.     

DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus.

The results of reciprocal DNA-DNA reassociation kinetics indicated that although the DNAs of human cytomegalovirus (CMV) strains Towne and AD169 shared approximately 90% of their nucleotide sequences, about 10% heterogeneity did exist. The implication was that, with respect to one another, the DNAs of CMV Towne and CMV AD169 contained unique nucleotide sequences. To obtain more direct evidence, 32P-labeled DNA of one virus strain was reassociated in the presence of excess unlabeled DNA of the heterologous virus strain. Those 32P-labeled DNA sequences remaining single stranded were separated from double-stranded DNA on hydroxyapatite columns and incubated with Southern blots containing XbaI restriction enzyme fragments of the homologous virus DNA. This approach not only enriched for nucleotide sequences unique to each strain of virus, but also provided for the identification of the restriction enzyme fragments in which the unique sequences were contained. The CMV Towne unique sequences were found in XbaI fragments A, C, G, L, N, and Q of CMV Towne DNA. The CMV AD169 unique sequences were found in XbaI fragments A, C, G, and J of CMV AD169 DNA. The possible significance of these data with respect to variation among other CMV isolates is discussed.     

Human cytomegalovirus binding to fibroblasts is receptor mediated.

The binding of radiolabeled human cytomegalovirus (HCMV) strain AD169 to human lymphocytes, lymphoblastoid cell lines, monocytes, and fibroblasts varied over a 20-fold range. Since maximum binding was observed with human foreskin fibroblasts (HFF), interactions of radiolabeled HCMV with this cell type were analyzed quantitatively. Binding of HCMV to HFF at 4 degrees C was specific and saturable; at low viral inputs specific binding averaged 16.4% of input and nonspecific binding was less than 1% of input. Binding curves yielded single-component linear Scatchard plots indicating an average Kd of 1.1 nM and 5,262 available virus-binding sites per cell. A two-component Scatchard curve was obtained at 37 degrees C and reflected viral internalization, since it could be converted to a single-component curve by the use of paraformaldehyde-fixed cells. HCMV strain Towne was found to bind to the receptor used by HCMV strain AD169 with similar affinity. HCMV failed to bind to protease-treated HFF or to HFF grown in the presence of inhibitors of glycosylation. Sialic acid residues, however, were not found to be important in binding. These data indicate that a single type of molecule, likely a glycoprotein, on the surface of HFF serves as a specific receptor for the virus.     

Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.     

UL146 variability among clinical isolates of human cytomegalovirus from Japan

Human cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.     

Human cytomegalovirus UL145 gene is highly conserved among clinical strains

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b') present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes,which are absent in the highly passaged laboratory strain AD169. One of these genes,UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene,the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9 -100% and 96.6-100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved,comprising the protein kinase C phosphorylation motif (PKC)and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.     

Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b'' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.     

Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo(BAC) virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo(BAC), like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo(BAC)) retained its virulence. The 15-kb segment was then deleted from Toledo(BAC), and the resulting virus, Toledo(Delta15kb), was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo(Delta15kb) had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo(Delta15kb) genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.     

Human retinal and brain cell lines: A model of HCMV retinitis and encephalitis

Although HIV is accepted as the etiologic agent in AIDS, other factors have been implicated in accelerating the disease. Human cytomegalovirus (HCMV) in particular has been implicated as a cofactor in the progression from AIDS-related complex (ARC) to AIDS. HCMV infection of the central nervous system (CNS) (brain, retina) has been reported in at least 50% of AIDS patients, and has been implicated in producing encephalitis and sight-threatening retinitis. HCMV exhibits strict species specificity and animal models for human HCMV are conspicuous by their absence. We have developed a human brain cell line (mixed glial/neuronal) and a multipotential human retinal precursor cell line (neuronal in nature). We have tested the suitability of these cell lines as models for the study of HCMV infectibility. In this study, we report that these cell lines are optimal for the study of HCMV infectibility and pathogenesis in tissues of neural origin and appropriate to study HIV-HCMV interaction. Immortalized human brain and retinal cell lines were infected with a laboratory strain of HCMV (AD 169, Towne) at a multiplicity of infection moi (1-5) and viral infectibility and cell specificity monitored by: (a) phenotypic analysis (multinucleate cells, syncytium formation, etc.), (b) antigen expression (IE, E, late) by immunohistochemistry, Western blot analysis, (c) presence of viral particles by TEM, and (d) expression of indicator plasmids (HIV-LTR-CAT). We report that both human retinal and brain cell lines are permissive for HCMV infectibility. Cell specificity was not seen; both cells expressing glial/neuronal cell markers were positive for the presence of HCMV early/late antigens. Formation of multinucleate giant cells with nuclear inclusion bodies and syncytia were seen. Productive viral infection was confirmed by the ability of cell-free supernatant from the third passage of infected cells to produce pathogenicity and express viral particles, when added to fresh cultures. Using indicator plasmids, HIV-LTR, and CAT, we have shown that HIV and HCMV interact at the cellular level. We have also shown that HIV production in retinal and brain cell lines transfected with cloned HIV was enhanced by HCMV-IE genes. We did not see any differences in HCMV. AD 169, Towne isolate, and data from both strains is presented in this paper. This model could prove extremely useful for the study of cell specificity/cellular and molecular interaction between HIV/HCMV and to test antiviral therapies.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.