Extended genotypic evaluation and comparison of twenty-two cases of lethal EEHV1 hemorrhagic disease in wild and captive Asian elephants in India.

Thirteen new lethal cases of acute hemorrhagic disease (HD) with typical histopathogical features were identified in young Asian elephants (Elephas maximus indicus) in India between 2013 and 2017. Eight occurred amongst free-ranging wild herds, with three more in camp-raised orphans and two in captive-born calves. All were confirmed to have high levels of Elephant Endotheliotropic Herpesvirus type 1A (EEHV1A) DNA detected within gross pathological lesions from necropsy tissue by multi-locus PCR DNA sequencing. The strains involved were all significantly different from one another and from nine previously described cases from Southern India (which included one example of EEHV1B). Overall, eight selected dispersed PCR loci totaling up to 6.1-kb in size were analyzed for most of the 22 cases, with extensive subtype clustering data being obtained at four hypervariable gene loci. In addition to the previously identified U48(gH-TK) and U51(vGPCR1) gene loci, these included two newly identified E5(vGPCR5) and E54(vOX2-1) loci mapping far outside of the classic EEHV1A versus EEHV1B subtype chimeric domains and towards the novel end segments of the genome that had not been evaluated previously. The high levels of genetic divergence and mosaic scrambling observed between adjacent loci match closely to the overall range of divergence found within 45 analyzed North American and European cases, but include some common relatively unique polymorphic features and preferred subtypes that appear to distinguish most but not all Indian strains from both those in Thailand and those outside range countries. Furthermore, more than half of the Indian cases studied here involved calves living within wild herds, whereas nearly all other cases identified in Asia so far represent rescued camp orphans or captive-born calves.

Clinico-pathologic features of fatal disease attributed to new variants of endotheliotropic herpesviruses in two Asian elephants (Elephas maximus).

The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.

Modern evolutionary history of the human KSHV genome.

The genomes of several human herpesviruses, including Kaposi sarcoma (KS) herpesvirus (KSHV), display surprisingly high levels of both genetic diversity and clustered subtyping at certain loci. We have been interested in understanding this phenomenon with the hope that it might be a useful diagnostic tool for viral epidemiology, and that it might provide some insights about how these large viral genomes evolve over a relatively short timescale. To do so, we have carried out extensive PCR DNA sequence analysis across the genomes of 200 distinct KSHV samples collected from KS patients around the world. Here we review and summarize current understanding of the origins of KSHV variability, the spread of KSHV and its human hosts out of Africa, the existence of chimeric genomes, and the concept that different segments of the genome have had different evolutionary histories.

Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PML-mediated transcriptional repression.

Human cytomegalovirus (HCMV) major immediate-early protein IE1 is an abundant 72-kDa nuclear phosphoprotein that is thought to play an important role in efficient triggering of the lytic cycle, especially at low multiplicity of infection. The best-known properties of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML oncogenic domains [PODs] or nuclear domain 10 [ND10]), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes. Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1. In this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells, as well as in in vitro assays, with up to 30% of the protein representing the covalently conjugated 90-kDa form in stable U373/IE1 cell lines. Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs. Surprisingly, unlike PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast two-hybrid assays, suggesting that some additional unknown intranuclear cofactors must play a role in IE1 sumoylation. Interestingly, stable expression of either exogenous PML or exogenous Flag-SUMO-1 in U373 cell lines greatly enhanced both the levels and rate of in vivo IE1 sumoylation during HCMV infection. Unlike the disruption of PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the disruption of PODs by HCMV IE1 proved not to involve proteasome-dependent degradation of PML. We also demonstrate here that the 560-amino-acid PML1 isoform functions as a transcriptional repressor when fused to the GAL4 DNA-binding domain and that wild-type IE1 inhibits the repressor function of PML1 in transient cotransfection assays. Furthermore, both IE1(1-346) and IE1(L174P) mutants, which are defective in displacing PML from PODs, failed to inhibit the repression activity of PML1, whereas the sumoylation-negative IE1(K450R) mutant derepressed as efficiently as wild-type IE1. Taken together, our results suggest that proteasome-independent disruption of PODs, but not IE1 sumoylation, is required for efficient IE1 inhibition of PML-mediated transcriptional repression.

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures.

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.

Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9.

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

Origin-independent assembly of Kaposi's sarcoma-associated herpesvirus DNA replication compartments in transient cotransfection assays and association with the ORF-K8 protein and cellular PML.

Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primase-associated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.

Morphogenesis of HHV8 in primary human dermal microvascular endothelium and primary effusion lymphomas.

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposure to TPA or butyrate of H HV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even before viral nucleocapsids (NC) were visible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.

A new primary effusion lymphoma-derived cell line yields a highly infectious Kaposi's sarcoma herpesvirus-containing supernatant.

A primary effusion lymphoma (PEL) cell line, JSC-1, that yields highly infectious Kaposi's sarcoma herpesvirus (KSHV) supernatants was established from the ascitic fluid of a human immunodeficiency virus-positive patient. Flow cytometry showed strong expression of CD45 and lambda light-chain restriction. Southern blot hybridization showed immunoglobulin heavy-chain gene rearrangements in the tumor and the resultant cell line consistent with B-cell lineage. Expression of viral genes was assessed by reverse transcription-PCR and immunohistochemistry. Only latent Epstein-Barr virus (EBV) gene expression was detected, and this was at a low level. In contrast, lytic and latent KSHV gene expression were detected. Tetradecanoyl phorbol acetate and butyrate upregulated KSHV lytic expression, but not EBV lytic expression. Viral supernatant from JSC-1 was much more efficient at infecting primary human dermal microvascular endothelial cells (DMVECs) with KSHV than supernatants from BC-3 or BCP-1 PEL cell lines. Quantitation of viral yields produced by the PEL lines showed at least 2 orders of magnitude more DNase I-resistant KSHV DNA in the JSC-1 supernatant compared to BC-3 or BCP-1 supernatants. KSHV infection in DMVECs was associated with a change from a cobblestone to a spindle shape, LANA expression, and an increased number of mitoses.

Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection.

In human cytomegalovirus (HCMV) infection, both of the major immediate-early proteins IE1(IE68, UL123) and IE2(IE86, UL122) target to PML protein-associated nuclear bodies known as PODs or ND10 at very early times after infection. IE1 causes a redistribution of both PML and IE1 from the PODs into a nuclear diffuse form, whereas IE2 initially localizes adjacent to PODs but later associates with viral DNA replication compartments. The peripheries of PODs are also believed to be sites for initiation of both viral IE transcription and DNA replication. However, because IE1 is nonessential at high multiplicity of infection (m.o.i.) in HF cells, the exact role of these processes in viral infection has been enigmatic. Therefore, we investigated the effects of overexpression of PML in the presence or absence of IE1 on the intranuclear distribution of IE2 and formation of viral DNA replication compartments, as well as on the levels of delayed-early and late viral transcription and protein accumulation. Infection with wild-type HCMV(Towne) and the IE1-deleted derivative HCMV(CR208), which fails to disrupt PODs, was compared in a pair of related astrocytoma/glioblastoma cell lines, the U373-Neo control and a variant U373-PML that constitutively overexpresses PML(560) in much larger than normal PODs. IFA studies on the localization patterns for IE1, IE2, and PML showed that, although the numbers of IE2-positive cells were not significantly reduced in either the wild-type virus-infected U373-PML cell line or in DeltaIE1-infected control cells, POD disruption by IE1 in wild-type virus infection was delayed by up to 6 h in U373-PML cells compared to control cells. Furthermore, there was considerable enhancement of IE2 colocalization with PODs in Delta IE1-infected U373-PML cells. Formation of viral DNA replication compartments in the U373-PML cell line was also greatly delayed, measured at fivefold lower after wild-type virus infection and 12-fold lower after infection with Delta IE1 than in the control cell line at 48 h at an m.o.i. of 1.0. The levels of representative early and late viral proteins detected by Western blotting were suppressed by fivefold and 22-fold at 24 and 72 h, respectively, in the U373-PML cell line, even with high m. o.i. wild-type HCMV infection. Decreased viral protein levels also occurred when control cells were infected with the Delta IE1 virus and these two effects were additive in the U373-PML cell line. Similarly, when U373-PML cells were infected with recombinant HCMV expressing an extragenic luciferase reporter gene under the control of viral early (Pol) or late (pp28) promoters, their transcriptional activation was reduced up to fivefold at both high and low m.o.i. compared to that of the control cells. Overall, these results suggest that POD disruption by IE1 and subsequent redistribution of both PML and IE1 at very early times after infection may play an important role in the efficient utilization of cellular transcription and replication machinery by HCMV and contribute to rapid progression of the HCMV lytic cycle.

Clinical and pathological findings of a newly recognized disease of elephants caused by endotheliotropic herpesviruses.

The unique clinical and pathological findings in nine Asian (Elephas maximus) and two African (Loxodonta africana) elephants from North American Zoos with a highly fatal disease caused by novel endotheliotropic herpesviruses are described. Identification of the viruses by molecular techniques and some epidemiological aspects of the disease were previously reported. Consensus primer polymerase chain reaction (PCR) combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and the second in African elephants. Disease onset was acute, with lethargy, edema of the head and thoracic limbs, oral ulceration and cyanosis of the tongue followed by death of most animals in 1 to 7 days. Pertinent laboratory findings in two of three clinically evaluated animals included lymphocytopenia and thrombocytopenia. Two affected young Asian elephants recovered after a 3 to 4 wk course of therapy with the anti-herpesvirus drug famciclovir. Necropsy findings in the fatal cases included pericardial effusion and extensive petechial hemorrhages in the heart and throughout the peritoneal cavity, hepatomegaly, cyanosis of the tongue, intestinal hemorrhage, and ulceration. Histologically, there were extensive microhemorrhages and edema throughout the myocardium and mild, subacute myocarditis. Similar hemorrhagic lesions with inflammation were evident in the tongue, liver, and large intestine. Lesions in these target organs were accompanied by amphophilic to basophilic intranuclear viral inclusion bodies in capillary endothelial cells. Transmission electron microscopy of the endothelial inclusion bodies revealed 80 to 92 nm diameter viral capsids consistent with herpesvirus morphology. The short course of the herpesvirus infections, with sudden deaths in all but the two surviving elephants, was ascribed to acute cardiac failure attributed to herpesvirus-induced capillary injury with extensive myocardial hemorrhage and edema.

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses.

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10).

During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation.

Heterogeneity of viral IL-6 expression in HHV-8-associated diseases.

In order to characterize the expression of the viral interleukin-6 (vIL-6) homologue in various human herpesvirus 8 (HHV-8)-associated diseases, in situ hybridization and immunohistochemistry were applied to formalin-fixed specimens. These assays showed consistent expression of vIL-6 in primary effusion lymphomas and in a case of human immunodeficiency virus (HIV)-associated lymphadenopathy with a Castleman's disease-like appearance. In contrast, Kaposi's sarcoma specimens showed marked differences among specimens. In a consecutive series of specimens from the Johns Hopkins archives, vIL-6 expression was demonstrated in one of 13 cases. However, among 7 specimens selected from the AIDS Malignancy Bank because of their high levels of the T1.1 lytic transcript and virion production, vIL-6 expression was consistently demonstrated in infiltrating mononuclear cells and occasional spindle-shaped cells. Thus vIL-6 expression in clinical specimens correlates with other measures of the lytic viral cycle. Both assays generally give congruent results and are consistent with the possibility that vIL-6 expression plays a role in the pathogenesis of a variety of HHV-8-associated diseases.

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

Comparison of genetic variability at multiple loci across the genomes of the major subtypes of Kaposi's sarcoma-associated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame K15 alleles at the right-hand end.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0. 35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized "orphan" region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.

KSHV strains: the origins and global spread of the virus.

Nucleotide sequence analysis at five distinct loci across the 140, 000 bp genomes of more than 60 KSHV samples from KS and PEL tumors from North America, Africa, the Middle East, Asia and the Pacific revealed that they cluster into four major subtypes (A, B, C and D) that have close associations with the geographic and ethnic background of the patients. In particular, the ORF-K1 protein subtypes encoded at the extreme LHS of the genome display up to 30% amino acid variability resulting from 85% non-synonymous nucleotide substitution rates. In addition, two alternative highly diverged forms of the complex spliced ORF-K15 gene (P or M) map at the extreme RHS of the genome and are essentially unlinked to the ORF-K1 genotypes. We conclude that: (1) KSHV is an ancient human virus with several major subtypes that reflect the migrationary divergence of modern human populations over the past 35,000-60,000 years; (2) the novel immunoglobulin receptor-like signal transducing protein ORF-K1 is subject to unusually strong biological selective pressures; and (3) a minority of KSHV genomes have undergone recombination events with a related virus producing two different alleles of the ORF-K15 latency membrane protein.

High-level variability in the ORF-K1 membrane protein gene at the left end of the Kaposi's sarcoma-associated herpesvirus genome defines four major virus subtypes and multiple variants or clades in different human populations.

Infection with Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is common in certain parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere, except in AIDS patients. Nevertheless, HHV8 DNA is found consistently in nearly all classical, endemic, transplant and AIDS-associated KS lesions as well as in some rare AIDS-associated lymphomas. The concept that HHV8 genomes fall into several distinct subgroups has been confirmed and refined by PCR DNA sequence analysis of the ORF-K1 gene encoding a highly variable glycoprotein related to the immunoglobulin receptor family that maps at the extreme left-hand end of the HHV-8 genome. Among more than 60 different tumor samples from the United States, central Africa, Saudi Arabia, Taiwan, and New Zealand, amino acid substitutions were found at a total of 62% of the 289 amino acid positions. These variations defined four major subtypes and 13 distinct variants or clades similar to those found for the HIV ENV protein. The B and D subtype ORF-K1 proteins differ from the A and C subtypes by 30 and 24%, respectively, whereas A and C differ from each other by 15%. In all cases tested, multiple samples from the same patient were identical. Examples of the B subtype were found almost exclusively in KS patients from Africa or of African heritage, whereas the rare D subtypes were found only in KS patients of Pacific Island heritage. In contrast, C subtypes were found predominantly in classic KS and in iatrogenic and AIDS KS in the Middle East and Asia, whereas U.S. AIDS KS samples were primarily A1, A4, and C3 variants. We conclude that this unusually high diversity, in which 85% of the nucleotide changes lead to amino acid changes, reflects some unknown powerful biological selection process that has been acting preferentially on this early lytic cycle membrane signalling protein. Two distinct levels of ORF-K1 variability are recognizable. Subtype-specific variability indicative of long-term evolutionary divergence is both spread throughout the protein as well as concentrated within two 40-amino-acid extracellular domain variable regions (VR1 and VR2), whereas intratypic variability localizes predominantly within a single 25-amino-acid hypervariable Cys bridge loop and apparently represents much more recent changes that have occurred even within specific clades. In contrast, numerous extracellular domain glycosylation sites and Cys bridge residues as well as the ITAM motif in the cytoplasmic domain are fully conserved. Overall, we suggest that rather than being a newly acquired human pathogen, HHV8 is an ancient human virus that is preferentially transmitted in a familial fashion and is difficult to transmit horizontally in the absence of immunosuppression. The division into the four major HHV8 subgroups is probably the result of isolation and founder effects associated with the history of migration of modern human populations out of Africa over the past 35,000 to 60,000 years.

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines.

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.