Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-jB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-jB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-jB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-jB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-jB as a regulatory mechanism of this signalling pathway.

bKaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) protein is constitutively expressed in all KSHV-infected cells, as well as in all forms of KSHV-associated malignancies. LANA1 is a multifunctional KSHV oncoprotein containing multiple repeat sequences that is important for viral episome maintenance and the regulation of cellular and viral gene expression. We characterize here multiple LANA1 isoforms and show that ϳ50% of LANA1 is naturally generated as N-terminally truncated shoulder proteins that are detected on SDS-PAGE as faster-migrating shoulder bands designated LANA1 S . Higher-molecular-weight LANA1 S isoforms initiate downstream at noncanonical sites within the N-terminal region, whereas lower-molecular-weight LANA1 S isoforms initiate downstream within the central repeat 1 domain. LANA1 S proteins lack an N-terminal nuclear localization signal motif, and some isoforms differ from full-length, canonical LANA1 by localizing to perinuclear and cytoplasmic sites. Although LANA1 has until now been assumed to be solely active in the nucleus, this finding indicates that this major KSHV oncoprotein may have cytoplasmic activities as well. KSHV overcomes its limited genetic coding capacity by generating alternatively initiated protein isoforms that may have distinct biological functions.

Kaposi's sarcoma associated herpesvirus (KSHV or human herpesvirus 8 [HHV-8]) is a gammaherpesvirus highly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease, an aggressive lymphoproliferative disorder. KSHV, like other gammaherpesvirus latently infects predominantly B-cells and endothelial cells. Infected cells retain the virus from one generation to the next existing as a multicopy circular episomal DNA in the nucleus, expressing a limited subset of viral genes. Of these latently expressed genes, LANA1, the latency associated nuclear antigen is highly expressed in all forms of KS-associated malignancies. Various studies so far show that LANA1 tethers the viral episomes to host chromosomes and binds to specific sites within and close to the TR elements contributing to the stable maintenance of the viral episomes in successive daughter cells. Anti-LANA1 intrabody strategies might represent a new therapeutic approach to treatment of KSHV infections, since LANA1 is regained for KSHV latency. In addition, the use of intrabodies can help drug development by mapping LANA1 inhibiting regions. We report development of several LANA1 specific single chain antibodies from immunized rabbits that can be expressed intracellularly, bind to LANA1 epitopes and can be used for functional KSHV studies on viral latency.

Kaposi's sarcoma (KS) was originally described in 1872 by the Hungarian dermatologist Moritz Kaposi as an idiopathic, multiple pigmented sarcoma of the skin. This type of KS, later classifi ed as classic KS, predominantly occurred in elderly people, particularly men of Mediterranean, Eastern European, or Jewish descent. A high occurrence of African or endemic KS was reported during the early 1950's in equatorial African countries, while during the 1960's KS emerged among immune-suppressed patients following organ transplantation. A tremendous increase in the incidence of KS was noticed during the early 1980's in parallel with the acquired immunodefi ciency syndrome (AIDS) epidemic, leading to a formal recognition of KS as an AIDS-associated malignancy. The geographic variations in the incidence of KS together with the relatively high occurrence of AIDS-KS, particularly among homosexual men, suggested the existence of a unique KS infectious agent (7, 8). In 1994 Chang and colleagues, amplifi ed two novel DNA sequences from an AIDS-associated KS lesion, by using a subtractive hybridization technique (representational difference analysis (RDA)). These sequences displayed homology to herpesviral capsid and tegument genes and led to the complete sequencing of the 165 Kbp viral genome of the newly discovered virus, Kaposi's Sarcoma-Associated Herpesvirus (KSHV) or human herpesvirus 8 (HHV-8). KSHV was established as being present in virtually all pathologically confi rmed KS specimens (11, 12). Treatment with highly active antiretroviral therapy (HAART) has been associated with a dramatic decline in the incidence of KS (31), so that AIDS-associated KS is uncommon overall in the Western world. Sub-Saharan Africa has the highest rates of infection (due mostly to the high rates of AIDS) with many countries experiencing sera-prevalence rates exceeding 60%, so that in certain African countries it is now the most common cancer in adult men, and the second most common in adult women (44). The incidence of KS among transplant recipients is proportional to the ethno-geographical prevalence of KSHV, ranging from 0.5% in most Western countries to 5.3% in Saudi Arabia (50). In addition to KS, which is a tumour of endothelial cell origin, several haematological predominantly B cell disorders are also associated with KSHV. The two most well characterized are AIDS-associated primary effusion lymphomas (PEL) and a subset of multicentric Castleman's disease (MCD), an aggressive lymphoproliferative disorder. PELs are monoclonal, non-Hodgkin's B cell lymphomas that are commonly co-infected with EBV. Cell lines established from PEL, unlike KS tumour explants, stably maintain viral episomes at high copy number (30-150 copies per cell) and are the source of virus for most virologic and serologic studies. Most studies on latency of gammaherpesviruses (EBV) used lymphoblastoid cell lines that were derived from lymphomas or were established by in vitro infection/immortalization of peripheral blood lymphocytes. In vitro immortalized cells lines...