Distinct biology of Kaposi's sarcoma-associated herpesvirus from primary lesions and body cavity lymphomas.

The DNA sequence for Kaposi's sarcoma-associated herpesvirus was originally detected in Kaposi's sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi's sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi's sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt's B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi's sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi's sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi's sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi's sarcoma-associated herpesvirus isolates from primary Kaposi's sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.

Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase.

Current molecular genetic strategies to inhibit productive human immunodeficiency virus type 1 (HIV-1) replication have involved the generation of gene products which provide intracellular inhibition of essential virally encoded proteins or RNA structures. A molecular strategy to excise proviral DNA from HIV-1-infected cells and render these cells virus free would provide an attractive direct antiviral strategy, providing a mechanism to remove viral genes from infected cells. The potential of such a molecular genetic intervention was examined by using the Cre-loxP recombination system. A recombinant HIV-1 clone, designated HIV(lox), that contains loxP within a nonessential U3 region of the long terminal repeats was synthesized. The loxP motif was maintained during replication of HIV(lox) in CEM cells, as demonstrated by reverse transcriptase PCR analyses of genomic RNA isolated from virions. Two different types of HIV-1-permissive cells, CEM cells and 293 cells expressing the CD4 glycoprotein, were transformed with a Cre expression vector which was shown to encode Cre DNA binding and recombinase activities. HIV(lox) infection of CEM or CD4+ 293 cells expressing Cre resulted in a substantial reduction in virus replication compared to control cells, and evidence for the presence of the expected excision product was found. Site-specific excision of HIV-1 can therefore be achieved by using this model system with acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.

Synthesis and processing of equine herpesvirus 1 glycoprotein D.

Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.

Expression of membrane-bound and secreted forms of equine herpesvirus 1 glycoprotein D by recombinant baculovirus.

Analyses of the synthesis and processing of recombinant full-length glycoprotein D of equine herpesvirus type 1 (EHV-1; gD392) or recombinant truncated gD (gD352) expressed in baculovirus-infected Sf9 cells revealed the following: (1) gD polypeptides encoded by both recombinant baculoviruses react with gD-specific antibodies including peptide-specific antiserum that neutralizes EHV-1 in a plaque reduction assay, (2) both the full-length recombinant gD392 and the truncated gD352 are expressed predominantly as gD species that contain high mannose-type oligosaccharides (55 kDa and 52 kDa, respectively), (3) both the full-length recombinant gD392 and the truncated gD352 are also expressed in lesser amounts as gD species that contain complex-type oligosaccharides (58 kDa and 55 kDa, respectively) as well as the unglycosylated forms of gD (43 kDa and 37 kDa, respectively), (4) flow cytometric analyses of cells expressing gD392 revealed that gD first appears on the cell surface at 24 h post infection; by 60 h, 95% of the cells express high levels of cell surface gD, (5) cells expressing gD352, in contrast to cells expressing gD392, secrete gD into the extracellular medium. This initial demonstration that immunoreactive EHV-1 glycoprotein D can be produced as a secreted polypeptide in the baculovirus system should provide reagents to assess the potential use of gD as a subunit vaccine in an animal model.

Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.

Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope.

Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.

The equine herpesvirus type 1 (EHV-1) homolog of herpes simplex virus type 1 US9 and the nature of a major deletion within the unique short segment of the EHV-1 KyA strain genome.

The DNA sequence of the short (S) genomic component of the equine herpesvirus type 1 (EHV-1)KyA strain has been determined recently in our laboratory. Analysis of a 1353-bp BamHI/PvuII clone mapping at the unique short/terminal inverted repeat (Us/TR) junction revealed 507 bp of Us and 846 bp of TR sequences as well as an open reading frame (ORF) that is contained entirely within the Us. This ORF encodes a potential polypeptide of 219 amino acids that shows significant homology to the US9 proteins of herpes simplex virus type 1 (HSV-1), EHV-4, pseudorabies virus (PRV), and varicella zoster virus (VZV). The US9 polypeptides of the two equine herpesviruses exhibit 50% identity but are twice as large as their counterparts in HSV-1, PRV, and VZV. All five US9 proteins are enriched for serine and threonine residues and share a conserved domain of highly basic residues followed by a region of nonpolar amino acids. DNA sequence and Southern blot hybridization analyses revealed that the Us of EHV-1 KyA differs from the Us of EHV-1 KyD and AB1 in that the ORFs encoding glycoproteins I and E and a unique 10-kDa polypeptide are deleted from the KyA genome. These data demonstrate that the predicted 10-kDa protein unique to EHV-1 is nonessential for replication in vitro and that EHV-1 glycoproteins I and E, like their equivalents in HSV-1 and PRV, are also nonessential. These findings and those reported previously by this laboratory and others reveal that the Us segment of EHV-1 comprises nine ORFs, two of which, US4 and 10-kDa ORF, are unique to EHV-1. The gene order of the Us is US2, protein kinase, gG, US4, gD, gI, gE, 10 kDa, and US9.

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.

Sequence analysis of a glycoprotein D gene homolog within the unique short segment of the EHV-1 genome.

DNA sequence analysis of one-third of the unique short (Us) segment of the equine herpesvirus type 1 (EHV-1) genome revealed an open reading frame (ORF) whose translated sequence exhibits significant homology to glycoprotein D of herpes simplex virus (HSV) types 1 and 2 and to pseudorabies virus (PRV) glycoprotein 50, the gD equivalent. The ORF of the EHV-1 gD homolog lies within the pSZ-4 BamHI/KpnI fragment (map units 0.865 to 0.872 and 0.869 to 0.884) and is capable of encoding a polypeptide of 385 amino acids (43,206 molecular weight). Analysis of the nucleotide sequence revealed a complete transcriptional unit including CAAT and TATA elements and signals for polyadenylation. The predicted protein exhibits features typical of a transmembrane protein: a hydrophobic N-terminal signal sequence followed by a probable cleavage site, four potential N-linked glycosylation sites, and a hydrophobic membrane-spanning domain near the carboxyl terminus followed by a charged membrane anchor sequence.

Physical structure and molecular cloning of equine cytomegalovirus DNA.

Restriction endonuclease (RE) mapping studies and molecular hybridization analyses were conducted to determine the molecular structure of the genome of equine cytomegalovirus (ECMV). The ECMV genome is a linear, double-stranded DNA with a molecular size of 126 +/- 0.6 MDa (189 kbp). A library of cloned BamHI, EcoRI, and HindIII fragments of the viral genome was used to construct RE maps. Individual 32P-labeled cloned DNA fragments were hybridized to Southern blots of viral genomic DNA digested to completion with BamHI, EcoRI, HindIII, or SalI. These analyses revealed that the ECMV genome consists of a 97-MDa unique long region which is bracketed by repeated sequences. At one terminus of the genome, a 21.3-MDa segment of repeated sequences with no apparent unique sequences was identified. At the other terminus, a 6-MDa unique region bracketed by 2.4-MDa repeat segments was identified. No submolar RE fragments were identified upon digestion of the ECMV genome with BamHI, EcoRI, HindIII, SalI, or other REs, including BclI, BglII, NruI, and XbaI. The genome possesses only two termini as judged by lambda exonuclease digestion and by T4 DNA polymerase end-labeling of the intact DNA followed by digestion with BamHI, EcoRI, HindIII, SalI, BclI, BglII, NruI, or XbaI. In addition, Southern blot analysis of DNA extracted from ECMV-infected rabbit kidney cells revealed that only one viral DNA fragment within the intracellular viral DNA pool contains fused genomic termini. Taken together, these observations indicate that the ECMV genome does not isomerize and suggest that the genome of ECMV may be unique among those of the herpesviruses and especially those of the betaherpesviruses (cytomegaloviruses) since it contains regions of extensive internal homology yet does not undergo isomerization. Lastly, the relatively small size of the viral genome indicates an evolutionary diversification among the cytomegaloviruses.