Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus

The genetic analysis of human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma-associated virus, has been hampered by severe difficulties in producing infectious viral particles and modifying the viral genome. In this article, we report the successful cloning of the HHV8 complete genome onto a prokaryotic F-plasmid replicon which allows the propagation of the recombinant viral DNA in Escherichia coli. The insertion of the F-plasmid into the HHV8 genome interrupts the ORF56 gene, whose expression product-by homology with the Epstein-Barr virus BSLF1 gene--is supposed to be necessary for lytic DNA replication. After introduction of the recombinant HHV8 DNA into 293 cells, early viral antigens are expressed, suggesting that spontaneous lytic replication is initiated. However, completion of the lytic program is prevented by the absence of the ORF56 protein, and a quasi-latent state is established. Upon reintroduction of the ORF56 viral gene, the block is overcome and infectious HHV8 virions are produced. As the recombinant HHV8 genome can be easily modified in E. coli, this experimental system opens the way to an extensive genetic analysis of other HHV8 functions.     

The human herpes virus 8-encoded viral FLICE-inhibitory protein induces cellular transformation via NF-kappaB activation

Infection with human herpes virus 8 (HHV8) has been associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. HHV8 encodes for a viral FLICE-inhibitory protein (vFLIP), designated K13, which resembles the prodomain of caspase-8 in structure and has been shown to protect cells against death receptor-induced apoptosis in vitro and in vivo. In this report, we present evidence that HHV8 vFLIP also possesses the unique ability of transforming Rat-1 and Balb/3T3 fibroblast cells, which is not shared by other vFLIPs. Rat-1 cells expressing HHV8 vFLIP form colonies in soft agar and form tumors in nude mice. The transforming ability of HHV8 vFLIP is associated with the activation of the NF-kappaB pathway and is blocked by molecular and chemical inhibitors of this pathway. Our results suggest that vFLIP K13 has activity beyond its role as an inhibitor of death receptor signaling and may play a causative role in the pathogenesis of HHV8-associated malignancies. Furthermore, inhibitors of the NF-kappaB pathway may have a role in the treatment of malignancies linked to HHV8 infection.     

Long-Term Infection and Transformation of Dermal Microvascular Endothelial Cells by Human Herpesvirus 8

Human herpesvirus 8 (HHV8) infects Kaposi's sarcoma (KS) spindle cells in situ, as well as the lesional endothelial cells considered to be spindle cell precursors. The HHV8 genome contains several oncogenes, suggesting that infection of endothelial and spindle cells could induce cellular transformation and tumorigenesis and promote the formation of KS lesions. To investigate the potential of HHV8 infection of endothelial cells to contribute to the development of KS, we have developed an in vitro model utilizing dermal microvascular endothelial cells that support significant HHV8 infection. In contrast to existing in vitro systems used to study HHV8 pathogenesis, the majority of dermal endothelial cells are infected with HHV8 and the viral genome is maintained indefinitely. Infection is predominantly latent, with a small percentage of cells supporting lytic replication, and latency is responsive to lytic induction stimuli. Infected endothelial cells develop a spindle shape resembling that of KS lesional cells and show characteristics of a transformed phenotype, including loss of contact inhibition and acquisition of anchorage-independent growth. These results describe a relevant model system in which to study virus-host interactions in vitro and demonstrate the ability of HHV8 to induce phenotypic changes in infected endothelial cells that resemble characteristics of KS spindle cells in vivo. Thus, our results are consistent with a direct role for HHV8 in the pathogenesis of KS.     

Human herpesvirus 8 seropositivity among sexually active adults in Uganda

IntroductionSexual transmission of human herpesvirus 8 (HHV8) has been implicated among homosexual men, but the evidence for sexual transmission among heterosexual individuals is controversial. We investigated the role of sexual transmission of HHV8 in a nationally representative sample in Uganda, where HHV8 infection is endemic and transmitted mostly during childhood.ResultsThe weighted prevalence of HHV8 seropositivity was 56.2%, based on 1302 seropositive individuals, and it increased significantly with age (P_trend<0.0001). In analyses adjusting for age, sex, geography, education, and HIV status, HHV8 seropositivity was positively associated with reporting two versus one marital union (OR:1.52, 95% CI: 1.17–1.97) and each unit increase in the number of children born (OR: 1.04, 95% CI: 1.00–1.08), and was inversely associated with ever having used a condom (OR: 0.64, 95% CI: 0.45–0.89). HHV8 seropositivity was not associated with HIV (P = 0.660) or with herpes simplex virus 2 (P = 0.732) seropositivity. Other sexual variables, including lifetime number of sexual partners or having had at least one sexually transmitted disease, and socioeconomic variables were unrelated to HHV8 seropositivity.ConclusionOur findings are compatible with the conclusion that sexual transmission of HHV8 in Uganda, if it occurs, is weak.     

唾液中人类疱疹病毒7型的分离与培养

目的：了解人群中人类疱疹病毒7型（HHIV－7）的感染情况,并对HHV－7进行分离培养、鉴定。方法：以聚合酶链反应（PCR）检测唾液中的HHV－7,再以巢式PCR及酶切加鉴别;分离其中2例标本中的病毒,以人脐带血单个核细胞（HCBMC）进行培养,再通过DNA序列测定来鉴定培养结果。结果：检测120例唾液标本有97例阳性,PCR阳性结果示186bp处有一荧光带,经ECORI酶切后成为126bp和60bp两条荧光带,巢式PCR结果示94bp处有一荧光谱,HCBMC可用于HHV－7的传代培养,唾液中分离的HHV－7与培养结果所测得的病毒序列一致。与Berneman分离测得的JI株结果相似,结论：人的唾液中HHV－7检出率高,HHV－7可在HCBMC中增殖。     

The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.     

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals

Human herpesvirus‐6 (HHV‐6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV‐6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4⁺ T cells and PBMCs but not CD8⁺ T cells from HHV‐6‐infected individuals was stimulated with HHV‐6‐infected cell lysates. Moreover, HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals have suppressive activity on naïve CD4⁺ T and CD8⁺ T cells from HHV‐6‐uninfected individuals. However, no increased proportion of CD4⁺ CD25⁺ Treg cells from HHV‐6‐infected individuals contributed to the suppressive activity of the HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals. Transwell experiments, ELISA and anti‐IL‐10 antibody blocking experiment demonstrated that IL‐10 may be the suppressive cytokine required for suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interleukin (IL)‐10 and IL‐4 further implicated the HHV‐6‐speciflc IL‐10‐producing CD4⁺ T cells in the suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interferon (IFN)‐γ demonstrated a decreased frequency of HHV‐6‐speciflc IFN‐γ‐producing CD4⁺ T, but not CD8⁺ T cells in HHV‐6‐infected individuals, indicating that it was the CD4⁺ Th1 responses in HHV‐6‐infected individuals that were selectively impaired. Our findings indicated that HHV‐6‐specific IL‐10‐producing CD4⁺ T cells from HHV‐6‐infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL‐10 production, with a few cells expressing IFN‐γ, but none expressing IL‐4. These cells may play an important role in latent HHV‐6 infection.     

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.     

Identification of antigenic proteins encoded by human herpesvirus 8 and seroprevalence in the general population and among patients with and without Kaposi's sarcoma

To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathione S-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1. 4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA-positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay.     

Reduced seroprevalence of Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 8 (HHV8), related to suppression of Anopheles density in Italy

In two formerly malarious parts of Italy, age-related seroprevalence rates of Kaposi's sarcoma-associated herpesvirus [human herpesvirus 8 (KSHV/HHV8)] were determined from local blood donors and correlated with periods of vector control during anti-malaria campaigns. In Veneto, decreased KSHV/HHV8 seroprevalence in the 1951-1955 birth cohort coincides with the peak of DDT house-spraying. In Sardinia, where larviciding augmented indoor DDT-spraying, a significant drop of KSHV/HHV8 seroprevalence between 1945 and 1950 and 1951-1955 birth cohorts (P = 0.0046) coincides with suppression of the malaria vector Anopheles labranchiae Falleroni (Diptera: Culicidae). These results are consistent with age-related association between KSHV/HHV8 seroprevalence rates in native/resident populations and the density of malaria vectors in Veneto and Sardinia. This example supports our 'promoter arthropod' hypothesis on the role of haematophagous insects [putatively blackflies (Simuliidae), sandflies (Phlebotominae) and biting midges (Ceratopogonidae), as well as mosquitoes] when their bites induce hypersensitivity and immunosuppression, potentiate KSHV/HHV8 transmission via human saliva (when insect bite lesions are licked by another person whose saliva carries the virus) and may facilitate Kaposi's sarcoma.     

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.     

HHV 8 seroprevalence and transmission within Albanian family groups

The recently discovered Human Herpesvirus 8 (HHV 8) is associated with all clinical forms of Kaposi's sarcoma. While early research suggested that the virus was transmitted sexually and that it was present only in KS patients, more recent studies seem to show that infection with the virus is more common than once thought, presenting differing distribution patterns in different geographical areas. In this study we analyze seroprevalence and transmission of HHV 8 in a sample of 86 family groups from Albania. Participants were selected among families requesting routine pre-expatriation medical examinations at the Poliambulatorio Padre "L. Monti" in Tirana. Specimens were collected from 180 healthy individuals and tested for the presence of a specific antibody. Antibody anti-HHV-8 detection was performed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). The study found an overall rate of HHV 8 seroprevalence of 20.0%. In 4.5% of couples the male and female were both positive, in 30.2% at least one partner was positive; (in 17.4% only the male was positive; in 12.8% only the female). These results support the hypothesis that HHV 8 spreads via multiple transmission routes.     

Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [(3)H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K(m) for thymidine (dThd) of approximately 33.2 microM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK(-) cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [(3)H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.     

Is antiviral therapy in gastroduodenal pathology necessary?

Mucous membrane of the gastroduodenal region of 104 patients with different pathologies (gastritis and erosive ulcerative alterations) was investigated by polymerase chain reaction (PCR). Examination of the biopsy material revealed frequent occurrence of pathogenic strains of Helicobacter pylori (up to 80%) and Herpesviridae (EBV, HHV 6, HHV 7, HHV 8) in various combinations. In such cases it is necessary to use adequate antibacterial and antiviral therapy with individual choice of immunomodulators.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.