Non-Human Primate Model of Kaposi's Sarcoma-Associated Herpesvirus Infection

Since Kaposi''s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi''s sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.     

Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Open reading frame 45 (ORF45) of Kaposi''s sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.Kaposi''s sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus and the causative agent of several human cancers, including Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease (3, 6). Like all herpesviruses, KSHV has two alternative life cycles, a latent and a lytic cycle. During latency, only a few viral genes are expressed, and no progeny viruses are produced. Under appropriate conditions, latent viral genomes are activated, initiate lytic replication, and express a full panel of viral genes, in a process that leads to viral assembly, release of progeny virus particles, and de novo infection of naïve cells (3, 6). KSHV establishes latent infection in the majority of infected cells in cases of KS, primary effusion lymphoma, and multicentric Castleman''s disease, but lytic replications occur in a small fraction. The recurrent and periodic lytic cycles of KSHV are believed to play critical roles in viral pathogenesis (6, 7).Open reading frame 45 (ORF45) is a KSHV-encoded gene product that plays a critical role in the viral lytic cycle. It is an immediate-early protein and is also present in viral particles as tegument protein (26, 27, 30). Disruption of ORF45 has no significant effect on overall viral lytic gene expression or DNA replication in BAC36-reconstituted 293T cells induced with both tetradecanoyl phorbol acetate (TPA) and sodium butyrate together, but the ORF45-null mutant produces 5- to 10-fold fewer progeny viruses than the wild type and the mutant virus has dramatically reduced infectivity, suggesting that ORF45 plays important roles at both early and late stages of viral infection (29). In addition to its roles as a tegument component, which are possibly involved in viral ingress and egress processes, KSHV ORF45 interacts with cellular proteins and modulates the cellular environment. At least two such functions have been described. First, KSHV ORF45 inhibits activation of interferon regulatory factor 7 (IRF-7) and therefore antagonizes the host innate antiviral response (28). Second, KSHV ORF45 interacts with p90 ribosomal kinase 1 and 2 (RSK1/RSK2) and modulates the extracellular signal-regulated kinase/RSK signaling pathway, which is known to play essential roles in KSHV reactivation and lytic replication (12). All of these data suggest that KSHV ORF45 is a multifunctional protein.ORF45 is unique to the gammaherpesviruses; it has no homologue in the alpha- or betaherpesviruses. ORF45 homologues have been identified as virion protein components in other gammaherpesviruses, such as Epstein-Barr virus (EBV), rhesus rhadinovirus (RRV), and murine herpesvirus 68 (MHV-68), suggesting that certain tegument functions of ORF45 are conserved (2, 11, 18). ORF45 homologues differ in protein length. KSHV ORF45 is the longest, at 407 amino acids (aa); RRV, EBV, MHV-68, and herpesvirus saimiri (HVS) have proteins of 353, 217, 206, and 257 aa, respectively. The limited homologies lie mostly at the amino- and carboxyl-terminal ends. The middle portion of KSHV ORF45 diverges from those of its homologues. The homologues differ in subcellular localization. We and others have reported previously that KSHV ORF45 is found predominantly in the cytoplasm (1, 21, 28, 30), whereas ORF45 of MHV-68 is found exclusively in the nucleus (9). Recently, we found KSHV ORF45 also present in the nuclei of BCBL-1 cells in what resembled viral replication compartments, suggesting that ORF45 could shuttle into the nucleus (12).Nucleocytoplasmic trafficking of proteins across the nuclear membrane occurs through nuclear pore complexes. Small molecules of up to approximately 9 nm in diameter, corresponding to a globular protein of approximately 40 to 60 kDa, can in principle enter or leave the nucleus by diffusion through nuclear pores (15, 17, 24). Large molecules are transported with the aid of a related family of transport factors, importins and exportins, which recognize nuclear localization sequence (NLS)-containing or nuclear export sequence (NES)-containing proteins (15, 17, 23). CRM1 (exportin 1) has been identified as a common export receptor that recognizes human immunodeficiency virus Rev-like leucine-rich NES sequences and is responsible for the export of such NES-containing proteins (4, 5, 19, 22). CRM1-dependent nuclear export is specifically inhibited by a pharmacological compound, leptomycin B (LMB), that interacts with CRM1 and thus blocks such NES-mediated protein export (4).To understand the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we attempted to locate the signals that control its subcellular localization. In the research reported here, we identified a leucine-rich NES and an adjacent basic NLS in KSHV ORF45. We demonstrated that the regulated intracellular trafficking of ORF45, especially its translocation into the nucleus, is important for KSHV lytic replication.     

Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection.