Kaposi's Sarcoma-associated Herpesvirus Infected Cells Research Articles

MiR-769-3p inhibits cellular proliferation of KSHV-infected SH-SY5Y cells through targeting mTOR.

The infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the most common causes of death in AIDS patients. Our studies have found that KSHV can infect SH-SY5Y cells (named SK-RG) in vivo and mTOR was up-regulated, which results in remarkable enhancement of cell proliferation, migration. But the regulatory role of mTOR in KSHV infected neurons has not yet been fully elucidated. Here, we find that miR-769-3p is decreased in SK-RG cells, which can exert anti-KSHV effect through negatively regulating the expression of mTOR. The knockdown of mTOR or overexpress of miR-769-3p decreased the proliferation, migration ability and cell cycle related protein of SK-RG cells, and the expression of KSHV related genes. In contrast, activating mTOR function by 3BDO treatment weakened the cellular behaviors of miR-769-3p overexpressing cells. Meanwhile, overexpressed miR-769-3p and rapamycin showed a shared inhibition trend in the effects on cell proliferation and motility. Our data indicated that miR-769-3p can inhibit cell proliferation and migration by down regulating mTOR in KSHV infected SH-SY5Y cells, and can be a candidate molecule for anti-KSHV therapy.

Elevated iNOS and 3′-nitrotyrosine in Kaposi's Sarcoma tumors and mouse model

Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication.

Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. N6-methyladenosine (m6A) is one of the most abundant chemical signatures found in viral RNA genomes and virus-encoded RNAs. Here, we combined antibody-independent next-generation mapping with direct RNA sequencing to address the epitranscriptomic status of PAN RNA in KSHV infected cells. We showed that PAN m6A status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed selenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and ligation assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. By applying comprehensive proteomic approaches, we identified writers and erasers that regulate the m6A status of PAN, and readers that can convey PAN m6A phenotypic effects. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing (SHAPE-MaP) outlined local and global structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay that takes place between the cellular epitranscriptomic machinery and a specific viral RNA in the context of KSHV infected cells.

Lack of CD8+ T-cell co-localization with Kaposi's sarcoma-associated herpesvirus infected cells in Kaposi's sarcoma tumors.

Despite the close association between Kaposi’s sarcoma (KS) and immune dysfunction, it remains unclear whether tumor infiltrating immune cells (TIIC), by their absence, presence, or dysfunction, are mechanistically correlated with KS pathogenesis. Therefore, their potential capacity to serve as prognostic biomarkers of KS disease progression or control is unclear. Because epidemic-KS (EpKS) occurs with HIV-1 co-infection, it is particularly important to compare TIIC between EpKS and HIV-negative African endemic-KS (EnKS) to dissect the roles of HIV-1 and Kaposi Sarcoma-associated herpesvirus (KSHV) in KS pathogenesis. This cross-sectional study of 13 advanced KS (4 EnKS, 9 EpKS) patients and 3 healthy controls utilized single-color immunohistochemistry and dual-color immunofluorescence assays to characterize and quantify KSHV infected cells in relation to various TIIC in KS biopsies. Analysis of variance (ANOVA) and Mann-Whitney tests were used to assess differences between groups where P-values < 0.05 were considered significant. The abundance of KSHV infected cells was heterogeneous in KS biopsies. Despite the presence of T-cell chemoattractant chemokine CxCL-9 in biopsies, CD8+ T-cells were sparsely distributed in regions with evident KSHV infected cells but were readily detectable in regions devoid of KSHV infected cells (P < 0.0001). CD68+ (M1) macrophages were evenly and diffusely distributed in KS biopsies, whereas, the majority of CD163+ (M2) macrophages were localized in regions devoid of KSHV infected cells (P < 0.0001). Overall, the poor immune cell infiltration or co-localization in KS biopsies independent of HIV-1 co-infection suggests a fundamental tumor immune evasion mechanism that warrants further investigation.

Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs.

Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS). KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.

Cellular MicroRNA Let-7a Suppresses KSHV Replication through Targeting MAP4K4 Signaling Pathways

BackgroundKaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, the most common AIDS-related malignancy. The majority of KS tumor cells harbor latent KSHV virus but only a small percentage undergoes spontaneous lytic replication. Viral reactivation from latency is crucial for the pathogenesis and development of KS, but the cellular mechanisms underlying the switch between viral latency and replication are not well understood.MethodsThe level of let-7 miRNAs and MAP4K4 in KSHV infected 293T cells were quantified by real-time PCRs. Let-7 expression was silenced by the miRNA sponge technique. In let-7a transfected 293T cells, the expression of MAP4K4 was measured by real-time PCR and western blot. Luciferease expression was employed to examine the effect of let-7a on the 3’-untranslated region (UTR) of the MAP4K4 gene in 293T cells. Real-time PCR was used to quantify the KSHV copy numbers in BC-3 cells in which the expression of let-7a and/or MAP4K4 were altered. Finally, ERK, JNK and p38 protein production and their phosphorylation status were detected by western blots in let-7a or MAP4K4 transfected BCBL-1 cells.ResultsThe expression of microRNA let-7 was dramatically decreased in KSHV infected 293T cells, but that of MAP4K4 was increased significantly. Let-7a is physically associated with and targets the MAP4K4 3’UTR, and inhibits MAP4K4 expression at both mRNA and protein levels. MAP4K4 stimulates KSHV reactivation from latency, whereas let-7a inhibits the function of MAP4K4 by reversing the function of MAP4K4 on JNK, phospho-JNK and phospho-ERK1/2 levels.ConclusionOur results establish that let-7a specifically suppresses MAP4K4 expression, and further inhibits KSHV reactivation by interfering with the function of MAP4K4 on the MAPK pathway, highlighting let-7a as a potential treatment for KS.

A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

The Viral KSHV Chemokine vMIP-II Inhibits the Migration of Naive and Activated Human NK Cells by Antagonizing Two Distinct Chemokine Receptors

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56Dim CD16Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56Bright CD16Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56Dim CD16Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.

Suppressive Regulation of KSHV RTA with O-GlcNAcylation

BackgroundThe replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored.MethodsBy using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA.ResultsWe found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes.ConclusionsKSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.

Hypoxia Enhances the Phosphorylation and Cytotoxicity of Ganciclovir and Zidovudine in Kaposi's Sarcoma-Associated Herpesvirus–Infected Cells

Primary effusion lymphoma (PEL) is a rare B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL is poorly responsive to standard cytotoxic chemotherapy and portends a poor survival. Consequently, new effective treatment options are urgently needed. It is known that KSHV encodes two lytic genes, ORF36 (phosphotransferase) and KSHV ORF21 (thymidine kinase), which can phosphorylate ganciclovir and azidothymidine, respectively. Here, we have explored whether these genes can be used as therapeutic targets for PEL. PEL arises in pleural spaces and other effusions that provide a hypoxic environment. Based on Northern blot analysis, exposure of PEL cells to hypoxia up-regulated the expression of both ORF36 and ORF21. Using a newly developed nonradioactive reverse-phase high-performance liquid chromatography/mass spectrometry method to separate and quantify the phosphorylated forms of ganciclovir and azidothymidine, we found that PEL cells exposed to hypoxia produced increased amounts of the toxic triphosphates of these drugs. Moreover, we found that hypoxia increased the cell toxicity of ganciclovir and azidothymidine in PEL cells but had no significant effect on the herpesvirus-negative cell line CA46. These findings may have clinical applicability in the development of effective therapies for PEL or other KSHV-related malignancies.

Protein complexes associated with the Kaposi's sarcoma-associated herpesvirus-encoded LANA

Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA is known to modulate viral and cellular gene expression. We report here on some initial proteomic studies to identify cellular proteins associated with the amino and carboxy-terminal domains of LANA. The results of these studies show an association of known cellular proteins which support LANA functions and have identified additional LANA-associated proteins. These results provide new evidence for complexes involving LANA with a number of previously unreported functional classes of proteins including DNA polymerase, RNA helicase and cell cycle control proteins. The results also indicate that the amino terminus of LANA can interact with its carboxy-terminal domain. This interaction is potentially important for facilitating associations with other cell cycle regulatory proteins which include CENP-F identified in association with both the amino and carboxy-termini. These novel associations add to the diversity of LANA functions in relation to the maintenance of latency and subsequent transformation of KSHV infected cells.

Recruitment of the de novo DNA methyltransferase Dnmt3a by Kaposi's sarcoma-associated herpesvirus LANA

The Kaposi's sarcoma-associated herpesvirus LANA protein is expressed in all Kaposi's sarcoma-associated herpesvirus-infected cells, including the tumor cells of endemic and AIDS-associated Kaposi sarcoma, primary effusion lymphoma, and Castleman disease. LANA modulates cell gene expression, but the mechanisms of LANA-mediated transcriptional reprogramming are poorly understood. LANA-repressed cell genes were identified by using retroviral-transduced telomerase-immortalized microvascular endothelial cells. Transciptional repression of targeted genes was relieved by treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation in repression. We found that LANA coprecipitated with DNA methyltransferases (Dnmts) and recruited endogenous DNA methyltransferase activity from the cell extract. LANA preferentially relocalized Dnmt3a from the nuclear matrix into the chromatin fraction. Further, LANA associated with repressed cellular promoters, recruited Dnmt3a to DNA, and facilitated de novo promoter methylation of a down-regulated gene, cadherin 13 (H-cadherin). The data provide an example of promoter-specific epigenetic DNA modification through viral protein recruitment of de novo Dnmt activity.

Spectroscopic analysis of Kaposi's sarcoma-associated herpesvirus infected cells by Raman tweezers

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), is a tumor causing virus. KSHV is the cause of several disease conditions known as Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma. Cell culture supernatants from KSHV infected hematopoietic cells induced angiogenic tubule formation to a significantly greater extent than uninfected hematopoietic cells. Raman spectrum profiles were generated to differentiate the uninfected from KSHV infected cells. In general, profiles from all the hematopoietic cells shared similar peaks; however, the relative abundance of specific components varied significantly between the cells. Subsequent use of the multivariate analysis of the Raman spectra revealed significant differences between the uninfected and the KSHV infected cells. Taken together, this study reports the use of Raman tweezers to distinguish and analyze the biological relevance of KSHV infected cell signaling.

Constitutively Active K-cyclin/cdk6 Kinase in Kaposi Sarcoma–Associated Herpesvirus–Infected Cells

Kaposi sarcoma-associated human herpesvirus (KSHV) encodes K-cyclin, a homologue of D-type cellular cyclins, which binds cyclin-dependent kinases to phosphorylate various substrates. K-cyclin/cdk phosphorylates a subset of substrates normally targeted by cyclins D, E, and A. We used cells naturally infected with KSHV to further characterize the biochemical features of K-cyclin. We used immunoprecipitation with K-cyclin antibodies to examine the association of K-cyclin with cdk2, cdk6, p21Cip1, and p27Kip1 proteins in BC3 cells. We separated populations of BC3 cells enriched in cells in G1, S, or G2/M phases by elutriation and measured K-cyclin protein and the kinase activity of K-cyclin/cdk6 complexes. The half-life of K-cyclin and cyclin D2 proteins was determined by blocking protein synthesis with cycloheximide and measuring proteins in cell lysates by western blot analysis. We fused the entire K-cyclin sequence to the carboxyl-terminal sequence of cellular cyclin D that contains the PEST degradation sequence to produce K-cyclin/D2 and transfected K-cyclin/D2 into K-cyclin-negative cells to investigate the effect of the PEST sequence on K-cyclin's stability. Viral K-cyclin interacted with cyclin-dependent kinases cdk2, cdk4, and cdk6 and with the cyclin/cdk inhibitory proteins p21Cip1 and p27Kip1 in BC3 cell lysates. Unlike D-type cyclins, whose expression is cell cycle dependent, the level of K-cyclin was stable throughout the cell cycle, and the kinase associated with the K-cyclin/cdk6 complex was constitutively active. The half-life of K-cyclin (6.9 hours) was much longer than that of cellular cyclin D2 (0.6 hour) and that of K-cyclin/D2 (0.5 hour), probably because K-cyclin lacks the PEST degradation sequence present in D-type cyclins. The constitutive activation of K-cyclin/cdk complexes in KSHV-infected cells appears to result from the extended half-life of K-cyclin and may explain its role in Kaposi sarcoma.

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Transmission and disease association of Kaposi's sarcoma-associated herpesvirus: recent developments.

Kaposi's sarcoma-associated herpesvirus or human herpesvirus 8, common in sub-Saharan Africa and around the Mediterranean Sea but rare in most other countries, is known to be transmitted in childhood within families in endemic regions, and through sexual contacts among high-risk groups in Western countries. Nevertheless recent developments on other modes of transmission of the virus have been made during the last years and are summarized in this review. Furthermore, recent published disease associations are discussed. The last year has seen research addressing the question of parenteral transmission, sexual transmission through heterosexual contact, transmission of Kaposi's sarcoma-associated herpesvirus-infected cells from organ donors to recipient, as well as the first suggestion that host genetic factors may facilitate infection in childhood. Additional clinical manifestations of infection with the virus such as primary pulmonary hypertension and germinotropic lymphoproliferative disorder have been identified. Evidence of Kaposi's sarcoma-associated herpesvirus transmission other than between homosexual adults and during childhood - namely transmission through heterosexual contact or injection drug use - is growing although these issues are still incompletely analysed and far away from being fully understood. Despite our increasing knowledge on transmission and disease associations of the virus, implications on the clinical management of associated diseases and public health have to be further evaluated in the coming years.