Rhesus monkey rhadinovirus: a model for the study of KSHV.

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi sarcoma-associated herpesvirus (KSHV)-a gamma-2 herpesvirus and the etiologic agent of three malignancies associated with immunosuppression. In contrast to KSHV, RRV displays robust lytic-phase growth in culture, replicating to high titer, and therefore holds promise as an effective model for studying primate gammaherpesvirus lytic gene transcription as well as virion structure, assembly, and proteomics. More recently, investigators have devised complementary latent systems of RRV infection, thereby also enabling the characterization of the more restricted latent transcriptional program. Another benefit of working with RRV as a primate gammaherpesvirus model is that its efficient lytic growth makes genetic manipulation easier than that in its human counterpart. Exploiting this quality, laboratories have already begun to generate mutant RRV, setting the stage for future work investigating the function of individual viral genes. Finally, rhesus macaques support experimental infection with RRV, providing a natural in vivo model of infection, while similar nonhuman systems have remained resistant to prolonged KSHV infection. Recently, dual infection with RRV and a strain of simian immunodeficiency virus (SIV) has led to a lymphoproliferative disorder (LPD) reminiscent of multicentric Castleman disease (MCD)--a clinical manifestation of KSHV infection in a subset of immunosuppressed patients. RRV, in short, shows a high degree of homology with KSHV yet is more amenable to experimental manipulation both in vitro and in vivo. Taken together, these qualities ensure its current position as one of the most relevant viral models of KSHV biology and infection.

Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SIV-positive and SIV-negative rhesus macaques.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease. Studies on the biology and pathogenesis of KSHV have been limited by lack of efficient cell culture systems and lack of a suitable animal model for KS. Here we report on the experimental inoculation of SIV-positive and SIV-negative rhesus macaques with KSHV-infected PEL cells or KSHV preparations derived from PEL cells. Low levels of viral DNA could be detected in cultivated peripheral blood mononuclear cell of all animals, as well as in the bone marrow of one monkey that died from SAIDS. However, we were not able to detect KSHV-specific antibodies or transcripts, nor did we observe any symptoms clearly related to KSHV infection (e.g. KS or lympho-proliferative disease). Hence, although KSHV replicates in rhesus macaques at very low levels, this non-human primate host is unlikely to provide a useful animal model for disease.

Definition of an optimal cytotoxic T lymphocyte epitope in the latently expressed Kaposi's sarcoma-associated herpesvirus kaposin protein.

Cytotoxic T lymphocytes (CTL) recognize and kill virus-infected cells and contribute to immunologic control of viral replication. For many herpesviruses (e.g., Epstein-Barr and cytomegalovirus), virus-specific CTL responses can be readily detected in infected persons, but CTL responses against Kaposi's sarcoma-associated herpesvirus (KSHV) appear to be weak and remain poorly characterized. Using a human leukocyte antigen (HLA) binding motif-based epitope prediction algorithm, we identified 37 HLA-A*0201 binding peptides from 8 KSHV open-reading frames (ORFs). After in vitro stimulation of peripheral blood mononuclear cells from KSHV-infected persons, CTL responses against 1 peptide in the KSHV kaposin protein (ORF K12) were detected in 2 HLA-A*0201-positive subjects. The optimal CTL epitope was identified by HLA restriction analysis and peptide titration assays. These data describe a latent phase viral gene product targeted by CTL that may be relevant for KSHV immunopathogenesis.

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus.

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.

Capsid structure of Kaposi's sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus.

The capsid of Kaposi's sarcoma-associated herpesvirus (KSHV) was visualized at 24-A resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the "floor") and chimney-like external protrusions. Overlying the floor at trigonal positions are (alpha beta(2)) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the alpha-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.

Use of epidemiologically well-defined subjects and existing immunofluorescence assays to calibrate a new enzyme immunoassay for human herpesvirus 8 antibodies.

Agreement between assays for the detection of human herpesvirus 8 (HHV-8) antibodies has been limited. In part, this disagreement has been because assay calibration (i.e., differentiating positive from negative results) has not been done in a standardized fashion with reference to a wide spectrum of HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) persons. To describe the performance of an assay for HHV-8 antibodies more accurately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodies to define two groups: a group of 135 HHV-8-infected individuals (true positives), including Kaposi's sarcoma patients and those asymptomatically infected, and a group of 234 individuals with a high likelihood of being HHV-8 uninfected (true negatives). A new enzyme immunoassay (EIA), using lysed HHV-8 virion as the antigen target, was then developed. With the above true positives and true negatives as references, the sensitivity and specificity of the EIA associated with different cutoff values were determined. At the cutoff that maximized both sensitivity and specificity, sensitivity was 94% and specificity was 93%. When the EIA was used to test a separate validation group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were seropositive, versus 6% seropositivity in a group of children, women, and heterosexual men. It is proposed that the EIA has utility for large-scale use in a number of settings and that the calibration method described can be used for other assays, both to more accurately describe the performance of these assays and to permit more-valid interassay comparison.

Evidence for concurrent epidemics of human herpesvirus 8 and human immunodeficiency virus type 1 in US homosexual men: rates, risk factors, and relationship to Kaposi's sarcoma.

We examined human herpesvirus 8 (HHV-8) seroprevalence and seroincidence among 245 homosexual men from New York City (NYC) and Washington, DC (DC) who have been followed since 1982. An immunofluorescence assay measured antibodies to a latent HHV-8 nuclear antigen. Seroprevalence was 20.4% in 1982; seroincidence was approximately 15%/year during 1982-1983 but fell sharply thereafter. NYC men had a higher seroprevalence (odds ratio, 3.43; P<.001) and seroincidence (rate ratio, 2.13; P=.01) than DC men. Risk of Kaposi's sarcoma (KS) was increased in seropositive men (adjusted relative hazard, 3.58; P=.02). Among men who were seropositive for both human immunodeficiency virus type 1 and HHV-8, the 10-year cumulative risk of KS was 39%; time from coinfection to KS diagnosis ranged from 15 to 154 months (median, 63.5 months). This study shows an epidemic of HHV-8 among US homosexual men in the early 1980s that was associated with a high risk of developing KS.

Do viral chemokines modulate Kaposi's sarcoma?

Kaposi's sarcoma (KS) is an angiogenic tumor of mixed cellularity most commonly found in homosexual men infected with HIV. Both molecular and epidemiologic evidence has linked a newly described herpesvirus to this disease. This virus, Kaposi's sarcoma-associated herpesvirus (KSHV), encodes a number of cellular homologues, including two genes that share remarkable similarity to the human chemokine macrophage inhibitory factor-1 alpha. Recently, studies have begun to shed light on the roles these viral chemokines (vMIP-I and vMIP-II) may play in the complex pathogenesis of KS. The vMIP peptides may contribute to the formation of new blood vessels (neovascularization), inhibit infection by certain strains of HIV-1 and modify the cellular immune response.

Sexual transmission and the natural history of human herpesvirus 8 infection.

BACKGROUND: Although human herpesvirus 8 (HHV-8) has been suspected to be the etiologic agent of Kaposi's sarcoma, little is known about its seroprevalence in the population, its modes of transmission, and its natural history. METHODS: The San Francisco Men's Health Study, begun in 1984, is a study of a population-based sample of men in an area with a high incidence of human immunodeficiency virus (HIV) infection. We studied all 400 men infected at base line with HIV and a sample of 400 uninfected men. Base-line serum samples were assayed for antibodies to HHV-8 latency-associated nuclear antigen (anti-LANA). In addition to the seroprevalence and risk factors for anti-LANA seropositivity, we analyzed the time to the development of Kaposi's sarcoma. RESULTS: Anti-LANA antibodies were found in 223 of 593 men (37.6 percent) who reported any homosexual activity in the previous five years and in none of 195 exclusively heterosexual men. Anti-LANA seropositivity correlated with a history of sexually transmitted diseases and had a linear association with the number of male sexual-intercourse partners. Among the men who were infected with both HIV and HHV-8 at base line, the 10-year probability of Kaposi's sarcoma was 49.6 percent. Base-line anti-LANA seropositivity preceded and was independently associated with subsequent Kaposi's sarcoma, even after adjustment for CD4 cell counts and the number of homosexual partners. CONCLUSIONS: The prevalence of HHV-8 infection is high among homosexual men, correlates with the number of homosexual partners, and is temporally and independently associated with Kaposi's sarcoma. These observations are further evidence that HHV-8 has an etiologic role in Kaposi's sarcoma and is sexually transmitted among men.

Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus.

Over 85% of patients with Kaposi's sarcoma (KS) are seropositive for antibodies to the latency-associated nuclear antigen (LANA) expressed in B cell lines infected with Kaposi's sarcoma-associated herpesvirus (KSHV). The presence of antibodies to LANA strongly correlates with the risk of developing the disease. However, the identity of the protein(s) comprising LANA and the corresponding gene(s) has remained unclear. To identify potential latent gene candidates for LANA, we probed total RNA extracted from BCBL-1 cells (a B cell line latently infected with KSHV) using lambda clones that span the KSHV genome. One region encoding latent transcripts spanned KSHV open reading frames (orfs) 71 (K13), 72 (v-cyclin), and 73. Among these, however, only orf 73, when expressed in heterologous mammalian cell systems, reacted with KSHV antibody-positive human sera, resulting in a punctate nuclear staining pattern reminiscent of LANA in BCBL-1 cells. Furthermore, extracts from cells expressing the orf 73 protein product specifically blocked the binding of KS patient antibodies to LANA. Finally, seroreactivity with recombinant orf 73 protein exactly paralleled reactivity with classical LANA as expressed in BCBL-1 cells, both in KS patients and in other groups. Together, these data support the identification of KSHV orf 73 as the gene encoding the dominant immunogenic component of LANA.

Absence of detectable human herpesvirus 8 in the semen of human immunodeficiency virus-infected men without Kaposi's sarcoma.

The prevalence of human herpesvirus 8 (HHV-8)/Kaposi's sarcoma (KS)-associated herpesvirus was investigated in the semen of 99 human immunodeficiency virus (HIV)-infected men (median CD4 cell count, 357/mm3) by use of a polymerase chain reaction (PCR) assay capable of detecting <10 copies of HHV-8 DNA. Of the subjects, 95 (96%) self-identified as men who have sex with men (MSM), and 3 had a history of clinical KS. Seminal cell specimens were negative for HHV-8 in 98 subjects. None of the 26 without KS (27.1% of 96 tested) who were seropositive for HHV-8 by IFA for latency-associated nuclear antigens had HHV-8 detected in their semen. The only subject with any evidence for seminal HHV-8 DNA was seropositive for HHV-8 and had active KS. HHV-8 was detected in 10 (10.4%) of 96 peripheral blood mononuclear cell specimens. The prevalence of HHV-8 DNA by PCR in semen of HIV-infected MSM without KS is low.

Sensitivity of Kaposi's sarcoma-associated herpesvirus replication to antiviral drugs. Implications for potential therapy.

Using a cell line (termed BCBL-1) derived from a peripheral effusion (body cavity-based) lymphoma latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV), we recently reported the successful induction of KSHV replication in culture (Renne, R., W. Zhong, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. Ganem. 1996. Nat. Med. 2:342-346). Here we report the first use of this system for establishing the susceptibility of KSHV to available antiviral drugs. Latently infected BCBL-1 cells were induced to lytic replication with phorbol esters; such cells secrete large numbers of KSHV virions into the culture medium. We assayed the ability of the antivirals to block KSHV production, as measured by the release of encapsidated viral DNA. The results show that KSHV replication is insensitive to acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine) (50% inhibitory concentration [IC50] = 60-80 microM), but sensitive to ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl]guanine) (IC50 = 2.7-4 microM), foscarnet (trisodium phosphonoformate hexahydrate) (IC50 = 80-100 microM), and cidofovir (1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (IC50 = 0.5-1 microM).

Antibodies to human herpesvirus 8 in women and infants born in Haiti and the USA.

PIP: 146 HIV-1-positive pregnant women and 143 matched HIV-1-uninfected pregnant women participated in a study to assess the association of human herpes virus 8 (HHV8) and the development of Kaposi's sarcoma. 98 of the 189 evaluable infants born to the women were born to HIV-1-positive mothers; 26 of these 98 infants were infected with HIV-1. Maternal serum samples were available for all but 18. The sera were diluted and screened for antibodies to the HHV8 latency-associated nuclear antigens (LANA) expressed in the BCBL-1 cell line. Coded sera randomly included from 10 healthy blood donors were negative for anti-LANA, while sera from two patients with AIDS and Kaposi's sarcoma were positive. 12 of the 289 pregnant women were seropositive for anti-HHV8, a proportion consistent with other findings in high-risk non-pregnant women. HIV-1-infected Haitians have an increased risk of Kaposi's sarcoma. The present cohort included 91 women of Haitian origin, nine who were seropositive for anti-HHV8. The proportion of HHV8 seropositive women among Haitians is significantly higher than among other women. HHV8 seropositivity was not increased among women with HIV-1 infection and all 189 infants were seronegative for HHV8. These findings support the association between HHV8 infection and the risk of Kaposi's sarcoma.^ieng

The prevalence of serum antibody to human herpesvirus 8 (Kaposi sarcoma-associated herpesvirus) among HIV-seropositive and high-risk HIV-seronegative women.

OBJECTIVE: To determine the prevalence of human herpesvirus 8 (HHV-8) seropositivity among women who are known to be infected with human immunodeficiency virus type 1 (HIV-1) or who are at high risk for HIV infection. DESIGN: A cross-sectional and blinded study of the prevalence of serological reactivity to HHV-8 infection as determined by an indirect immunofluorescence assay using nuclei from cells latently infected with HHV-8. Data and specimens were collected at baseline assessments of a longitudinal natural history cohort study. SETTING: Four San Francisco Bay Area outpatient HIV specialty clinics participating in the cohort study. PATIENTS: A total of 387 participants in the Women's Interagency HIV Study whose HIV infection status was documented and serum was available in a local specimen repository. MAIN OUTCOME MEASURE: Serological reactivity to HHV-8. RESULTS: Serological reactivity to latent HHV-8 antigens was uncommon among study participants: 13 (3.4%) demonstrated serum antibodies. HHV-8 reactivity was more common among HIV-infected women; 12 (4.0%; 95% confidence interval [CI], 2.1%-6.9%) of the 302 HIV-infected participants vs 1 (1.2%; 95% CI, 0.03%-6.4%) of the 84 HIV-uninfected participants were seropositive for HHV-8, though the difference did not attain statistical significance (odds ratio=3.43; 95% CI, 0.49-148.6). Two of the HIV-infected women had Kaposi sarcoma lesions and both were seropositive. CONCLUSIONS: The prevalence of HHV-8 seropositivity among the group of HIV-infected women was dramatically lower than that recently reported among HIV-infected homosexual and bisexual men (30%-35%). This finding parallels the lower prevalence of Kaposi sarcoma among women in contrast to men with HIV infection. These data further extend the correlation of HHV-8 serological reactivity with risk of Kaposi sarcoma and are consistent with an important role for HHV-8 infection in development of Kaposi sarcoma.

The seroepidemiology of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus): distribution of infection in KS risk groups and evidence for sexual transmission.

Striking differences in Kaposi's sarcoma (KS) risk for AIDS patients who acquire HIV via homosexual activity and those whose HIV infections derive from blood product exposure suggest the presence of a sexually transmitted agent other than HIV in the development of KS. Using an immunofluorescence assay, we examined serum samples from 913 patients for the presence of antibody specific for infection by human herpesvirus 8 (HHV8), an agent whose genome is regularly found in KS tissue. The distribution of HHV8 seropositivity conforms to that expected for a sexually transmitted pathogen and tracks closely with the risk for KS development. Our data support the inference that this virus is the etiologic cofactor predicted by the epidemiology of KS.

Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture.

Kaposi's sarcoma (KS) is the leading neoplasm of AIDS patients, and HIV infection is known to be a major risk factor for its development. However, KS can occur in the absence of HIV infection and the risk of KS development varies widely even among HIV-infected patients, with homosexual men with AIDS being 20 times more likely to develop KS than AIDS-afflicted children or hemophiliacs. These and other data strongly suggest that a sexually transmitted agent or co-factor may be involved in KS pathogenesis. Recently, DNA sequences corresponding to the genome of a novel member of the herpesvirus family have been identified within AIDS-KS biopsies, and several reports indicate that these sequences are also present in all forms of HIV-negative KS. These and other findings suggest this new agent, referred to as KS-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), as a candidate for the putative etiologic cofactor. However, the role of this agent in KS remains hotly debated. Further progress in understanding its biology has been severely hampered by the lack of a cell culture system for virus growth. Here we report the development of a system for the lytic growth of this virus in a latently infected B cell line and present the first ultrastructural visualization of the virus. This system will facilitate the detailed study of the molecular biology of viral replication, the testing of antiviral drugs and the development of diagnostic tests for viral infection.

Correct in vivo splicing of the mouse immunoglobulin kappa light-chain pre-mRNA is dependent on 5' splice-site position even in the absence of transcription.

In transcripts from the rearranged mouse immunoglobulin kappa light-chain locus, the intron separating the variable (V) plus joining (J) exon from the constant (C) exon contains up to three additional J regions, each with a functional 5' splice site. Previously, HeLa cells transfected with DNA encoding kappa light chains have been shown to mimic kappa-producing lymphocytes in splicing exclusively to the upstream-most 5' splice site, whereas selectivity is lost when kappa transcripts containing two more J regions are incubated in HeLa cell or lymphocyte nuclear extracts. Here we demonstrate that the fidelity of in vivo splicing depends on neither V-J rearrangement, the instability of erroneously splicing transcripts, nor a hierarchy of J-region 5' splice site utilization. Analysis of the splicing of presynthesized kappa transcripts injected into Xenopus oocytes demonstrates the correct 5' splice-site selection is independent of transcription. Implications for in vitro studies of regulated splice-site pairing are discussed.

Accurate 5' splice-site selection in mouse kappa immunoglobulin light chain premessenger RNAs is not cell-type-specific.

In mature mouse B lymphocytes, immunoglobulin kappa light chain transcripts contain an intervening sequence separating the recombined variable (V) plus joining (J) exon from the distant constant (C) exon. After V-J recombination, this intervening sequence can include as many as three unused but very similar J-region 5' splice sites. Each of these sites is potentially functional if the gene is appropriately recombined. It is unclear how the splicing machinery distinguishes among these 5' splice sites, always choosing the most upstream site. We used synthetic transcripts of kappa gene sequences containing J3 and J4 in both the germ-line and the recombined configurations to study the pattern of 5' splice-site selection in vitro. We find that both HeLa cell and lymphocyte nuclear extracts fail to discriminate between the J3- and J4-region 5' splice sites. In contrast, after transfection into HeLa cells, similar kappa light chain transcripts are spliced correctly at the most upstream 5' splice site--that which is used in kappa-producing cells. We conclude that accurate 5' splice-site selection in the mouse kappa light chain is neither cell-type- nor species-specific. Potential mechanisms for this controlling step in gene expression are discussed.

Nerve growth factor-induced stimulation of alpha-aminoisobutyric acid uptake in PC 12 cells: evaluation of its biological significance.

In the clonal PC12 pheochromocytoma cell line, the observed effects of nerve growth factor (NGF) on the uptake rates of alpha-aminoisobutyric acid (AIB) depend upon the assay conditions employed. In orthodox uptake assays calling for serum removal prior to the addition of AIB, 50 ng/ml NGF causes a stimulation of uptake of 62% after 40 minutes and 46% after 24-hours exposure. However, serum stimulates AIB uptake to a similar extent and the effects of serum and NGF are not additive. An uptake assay which directly measures the AIB uptake experienced by PC12 cells undergoing NGF-induced morphological differentiation was therefore employed. When compared with control cells growing in serum-containing medium, NGF-induced differentiating PC12 calls experience (1) only a transient and modest increase in AIB uptake, and (2) a significant long-term decrease in AIB uptake under conditions optimal for differentiation. It is concluded that NGF-directed neurite outgrowth is not mediated by NGF effects on amino acid uptake via the A-system.