Retroviruses depend on the virally encoded IN proteins to facilitate stable insertion of their reverse-transcribed genomes into host cell chromosomes. INs recognize the attachment (att) sites at the ends of long terminal repeats (LTRs) in viral DNA to carry out two sequential enzymatic reactions. In the first reaction, referred to as 3= processing, IN removes dinucleotides from the 3= ends of viral DNA to expose the 3= OH groups attached to the invariant CA dinucleotides. In the second reaction, DNA strand transfer, IN inserts the processed 3= termini into opposing strands of the host chromosomal DNA via a transesterification mechanism (1, 2). Host cell enzymes complete the process by repairing the single-stranded gaps on both sides of integrated viral DNA. Consequently, the resulting provirus is flanked by short duplications of the target DNA sequences. The duplication size appears to be retroviral genus specific, being 5 bp for human immunodeficiency virus type 1 (HIV-1) and 4 bp for murine leukemia virus (MLV) (3-5). The terminal cleavage and strand transfer steps can be observed in vitro with purified recombinant retroviral IN and DNA substrates, demonstrating that IN alone is sufficient to carry out these reactions (3, 6).Retroviral IN consists of three structural domains (reviewed in reference 7). The N-terminal domain (NTD) contains the zinc binding HHCC motif, and a highly conserved catalytic core domain (CCD) contains the essential active site Asp, Asp, and Glu (D, D-35-E motif) residues, which are directly involved in the catalytic activities of IN. The C-terminal domain (CTD) is least conserved (8-11). Mounting evidence suggests that IN functions as a tetramer (12-15). Recent crystal structures of the prototype foamy virus (PFV) IN bound to its viral and host DNA substrates revealed that all three IN domains participate in tetramerization and interactions with viral DNA (16,17).Retroviral integration into cellular DNA does not occur in a random manner with respect to various genomic features (reviewed in reference 18). HIV-1 and other lentiviruses show a remarkable preference for integration within active transcription units (19). In contrast, MLV, a gammaretrovirus, preferentially integrates near transcription start sites and CpG islands, features that are largely avoided by HIV-1 (20, 21). The remaining retroviral genera show other, albeit far less contrasting, integration patterns (22). Integration site selection of HIV-1 and other lentiviruses was shown to depend on the cellular protein lens epithelium-derived growth factor (LEDGF) (reviewed in reference 23). The IN binding domain (IBD) located within the C-terminal region of LEDGF mediates its interactions with HIV-1 and other lentiviral . LEDGF associates with chromatin via its N-terminal PWWP domain, which selectively binds to nucleosomes containing H3 trimethylated on Lys36 (27, 28), an epigenetic mark associated with bodies of transcription units (29). In cells depleted of LEDGF/p75, HIV-1 integration and replication were significantly affect...

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNAbinding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.X-ray crystallography | gammaherpesvirinae | viral latency | DNA-binding protein | KSHV LANA K aposi sarcoma herpesvirus (KSHV) is the only known γ 2 -herpesvirus of concern to human health. Apart from its involvement in two lymphoproliferative disorders, KSHV plays a vital role in the development of Kaposi sarcoma, the most common form of cancer in patients with AIDS (1, 2). After a primary infection event, the virus establishes lifelong latent persistence in the nuclei of its host cells. A molecular key player in the establishment, maintenance, and regulation of KSHV latency is the latency-associated nuclear antigen (LANA).KSHV LANA contains 1,162 amino acids in the prototype strain and exerts functions in host cell survival, transcriptional control, latent viral replication, and stable episome segregation during mitosis (3,4). Its N-terminal domain is separated from its C-terminal domain by a large internal repeat region (5, 6). Although the N-terminal domain and the internal repeat region are predicted to be only poorly structured (7), the C-terminal domain comprises a stable 3D structure with a strong hydrophobic core (8, 9). LANA's C-terminal domain binds to the LANA binding sites (LBS) within the viral terminal repeats (TRs) in a sequence-specific manner (10, 11). Therefore, this domain is referred to as the DNA-binding domain (DBD). In contrast, the N terminus of LANA binds to nucleosome...

The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGASmediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.KSHV | cytoplasmic LANA | cyclic GMP-AMP synthase

Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA–mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus–host interactions during reactivation and may represent potential novel targets for therapeutic intervention.