HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus.

The latency-associated nuclear antigen (LANA-1) of Human Herpes Virus 8 (HHV-8), alternatively called Kaposi Sarcoma Herpes Virus (KSHV) is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin 1. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells 2. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3). The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2). The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid) region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.

High rate of proV47A selection in HIV-2 patients failing lopinavir-based HAART.

We describe the emergence of the proV47A mutation in three out of five HIV-2-infected individuals failing lopinavir/ritonavir-based HAART. The appearance of such mutated variants resulted in high levels of phenotypic resistance to lopinavir, cross-resistance to indinavir, amprenavir, and hypersusceptibility to saquinavir. A search in HIV-2 databases revealed that proV47A is present in 8.6% of protease inhibitor (PI)-experienced patients but absent in all PI-naive patients. Its selection may be a common mutational pathway for developing resistance to lopinavir/ritonavir in HIV-2.

Risk of selecting K65R in antiretroviral-naive HIV-infected individuals with chronic hepatitis B treated with adefovir.

Seven antiretroviral-naive HIV-infected individuals with chronic hepatitis B treated with adefovir for longer than 6 months were assessed. Using bulk population sequencing and a sensitive limiting dilution analysis, the selection of K65R or other resistance mutations did not occur in HIV, suggesting that adefovir can be confidently used as hepatitis B virus (HBV) therapy in HIV/HBV-co-infected patients who do not require antiretroviral therapy.

Brd2/RING3 interacts with a chromatin-binding domain in the Kaposi's Sarcoma-associated herpesvirus latency-associated nuclear antigen 1 (LANA-1) that is required for multiple functions of LANA-1.

Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episomal DNA, to modulate transcription, and to interact with Brd2/RING3. Similar to the recently described tethering of bovine papillomavirus E2 protein to host chromatin via Brd4/MCAP, Brd2/RING3, another member of the Brd family of chromatin-binding proteins, therefore interacts with a chromatin-binding region of another viral latent nuclear protein and could play a role in its multiple functions.

A Domain in the C-terminal region of latency-associated nuclear antigen 1 of Kaposi's sarcoma-associated Herpesvirus affects transcriptional activation and binding to nuclear heterochromatin.

The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.

Risk factors for human herpesvirus 8 infection in a cohort of drug users in the Netherlands, 1985-1996.

To elucidate the mode of human herpesvirus 8 (HHV-8) transmission in a population of Amsterdam drug users, HHV-8 seroprevalence and seroincidence were determined in 1179 drug users in the Amsterdam Cohort Studies (1985-1996). Risk factors for HHV-8 infection were examined. Serum samples were screened with an enzyme immunoassay by using HHV-8 lytic capsid (open-reading frame [ORF] 65) and latent nuclear (ORF73) antigens; positive results were confirmed by Western blot and immunofluorescence assay. Seroprevalence (men, 3.4%; women, 1.4%) and seroincidence (men, 0.08; women, 0.05/100 person-years) were low in this study. Infections with human immunodeficiency virus (HIV) type 1, hepatitis B virus (HBV), and hepatitis C virus (HCV), but not HHV-8, were associated with injection drug use (IDU). Independent risk factors for HHV-8 seropositivity were homosexual contacts and Mediterranean nationality for men and sexual contact with bisexual men, absence of a steady partner, and unprotected commercial sex for women. Unlike HIV-1, HBV, or HCV infection, HHV-8 infection is uncommon in Amsterdam drug users, as is HHV-8 transmission through IDU.