Identification and Characterization of Kaposi’s Sarcoma-Associated Herpesvirus K8.1 Virion Glycoprotein

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi’s sarcomas (KS), body cavity-based lymphomas (BCBL), and some forms of Castleman’s disease. Previous serological tests with KS patient sera have detected lytic-cycle polypeptides from KSHV-infected BCBL cells. We have found that these polypeptides are predominantly encoded by the K8.1 open reading frame, which is present in the same genomic position as virion envelope glycoproteins of other gammaherpesviruses. The cDNA of K8.1 from BCBL-1 cells was found to encode a glycosylated protein with an apparent molecular mass of 37 kDa. K8.1 was found to be expressed during lytic KSHV replication in BCBL-1 cells and was localized on the surface of cells and virions. The results of immunofluorescence and immunoelectron microscopy suggest that KSHV acquires K8.1 protein on its virion surface during the process of budding at the plasma cell membrane. When KSHV K8.1 derived from mammalian cells was used as an antigen in immunoblot tests, antibodies to K8.1 were detected in 18 of 20 KS patients and in 0 of 10 KS-negative control subjects. These results demonstrate that the K8.1 gene encodes a KSHV virion-associated glycoprotein and suggest that antibodies to K8.1 may prove useful as contributory serological markers for infection by KSHV.     

A Rhesus Macaque Rhadinovirus Related to Kaposi’s Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Encodes a Functional Homologue of Interleukin-6

The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi's sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6). To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathione S-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays. Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner. Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6. Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells. These results demonstrate that RRV encodes an accessory protein with IL-6-like activity.     

Identification of an Immunoreceptor Tyrosine-Based Activation Motif of K1 Transforming Protein of Kaposi’s Sarcoma-Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi’s sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.     

K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

The Kaposi's sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, approximately 50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman's disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.     

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

Kaposi''s sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi''s sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 Å. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8''s mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.Most if not all organisms with DNA genomes have mechanisms to ensure processive DNA synthesis. In bacteria, archaea, and eukaryotes, DNA polymerase subunits include a catalytic subunit and a processivity factor, often referred to as a “sliding clamp.” In these organisms, a clamp loader protein is required to assemble the processivity factor onto the DNA (27, 37). The bacterial sliding (beta) clamp is made up of homodimers of a subunit that comprises three structurally similar subdomains (26), whereas archaeal and eukaryotic proliferating cell nuclear antigen (PCNA) is composed of homotrimers that comprise two structurally similar subdomains (27, 37). For both of these clamps, the monomers assemble head-to-tail to form a closed homodimeric or homotrimeric ring, respectively, around the DNA. In these organisms, a clamp loader protein is required to efficiently load the clamp onto DNA, using an ATP-dependent process. Once loaded on DNA, the processivity factor is capable of binding directly to the DNA polymerase, conferring extended strand synthesis without falling off of the template (50).Herpesviruses encode their own DNA polymerases. However, unlike bacteria, archaea, and eukaryotes, herpesviruses do not encode clamp loaders to assemble their processivity factors onto the DNA. Yet, the accessory subunits of the herpesvirus DNA polymerases still associate with DNA with nanomolar affinity to enable long-chain DNA synthesis (9, 16, 23, 25, 29, 35, 44, 46, 53, 56). Human herpesviruses are divided into three classes, namely, the alpha-, beta-, and gammaherpesviruses, based on homologies found in their genomic organization as well as in protein sequences and function (45). Crystal structures have been determined for the processivity factor UL42 from the alphaherpesvirus herpes simplex virus type 1 (HSV-1) and for UL44 from the betaherpesvirus human cytomegalovirus (HCMV) (2, 3, 58). Despite having little if any sequence homology with processivity factors outside of their herpesvirus subfamily, these structures all share the “processivity fold” originally seen in the structure of the bacterial beta clamp (26). Interestingly, some of these processivity factors have a different quaternary structure. PCNA forms a head-to-tail trimeric ring (18, 27), HSV-1 UL42 is a monomer (10, 14, 16, 46, 58) equivalent to one-third of the PCNA complex, and HCMV UL44 is a head-to-head dimer in the form of a C-shaped clamp (2, 3, 9).Both HSV-1 UL42 and HCMV UL44 have a basic face that has been shown to be important for interacting with DNA (25, 35). In the case of dimeric HCMV UL44, the basic surface of each monomer faces inward, toward the center of the C clamp, and includes a basic loop, called the “gap loop,” that is thought to wrap around DNA (24). Recently the crystal structure of the bacterial beta clamp was determined in complex with DNA (15). In that structure, DNA was found to be located in the central pore of the clamp. Amino acid residues that interacted with DNA were in positions structurally homologous to those found on the positively charged faces of UL42 and UL44.UL42 and UL44 each also has a surface, facing away from the DNA binding face, that is important for interacting with the catalytic subunit of the viral DNA polymerase. Indeed, both of these proteins have been crystallized in complex with C-terminal peptides from their respective catalytic subunits, HSV-1 UL30 and HCMV UL54 (2, 58). Together with biochemical and mutational analyses, these crystal structures indicated that, although the details of the interaction are different, the catalytic subunit of the polymerase binds to a region including and in close proximity to a long loop that connects the N- and C-terminal subdomains, called the interdomain connector loop (32-34). The corresponding region of PCNA is also important for polymerase attachment and mediates the interactions of PCNA with many other cellular proteins (40). Both UL54 and UL30 were shown to attach to their respective subunits, UL44 and UL42, by way of their extreme C termini. The C-terminal residues responsible for this interaction correspond to amino acids that are not detectably conserved, either by sequence or by structure, among herpesvirus catalytic subunits. The HSV-1 UL30-UL42 interaction involves a groove to one side of the UL42 connector loop, with hydrophilic interactions being critical (58). The HCMV UL54-UL44 interaction involves a crevice near the UL44 connector loop, and hydrophobic interactions are crucial (2, 32, 33). Moreover, the HCMV UL44 crevice is on the opposite side of the connector loop with respect to the HSV-1 UL42 groove.Kaposi''s sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus, encodes a viral DNA polymerase catalytic subunit, Pol-8, and an accessory subunit, PF-8 (4, 7, 8, 29, 48, 57). PF-8 can bind to Pol-8 directly and specifically (8, 29) and is required for long-chain DNA synthesis in vitro (29). Similarly to UL44, PF-8 forms dimers in solution and when bound to DNA (9). Although it is likely that UL44 and PF-8 are the processivity factors for HCMV and KSHV, respectively, rigorous experiments demonstrating this have not been performed. However, for the sake of brevity and clarity, we will refer to these proteins as processivity factors.Here we present the crystal structure of PF-8 and show that PF-8 forms a head-to-head homodimer akin to UL44 but lacking the long gap loops which are thought to wrap around DNA. This suggests that PF-8 binds DNA differently than does UL44 or UL42. Because Pol-8 appears to lack a long, flexible C-terminal tail with a length comparable to those of other herpesvirus Pols, we expect the mode of binding of the catalytic subunit to be different as well. Based on structural data, information from homologs, and initial biochemical results, we were able to identify possible sites for interactions with DNA and Pol-8 and to propose a model for the simultaneous interaction of all three components of the complex. Further, the availability of crystal structures for all three herpesvirus classes provides new insights into comparative structure, function, and evolution.     

The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases

BLM and WRN, the products of the Bloom’s and Werner’s syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3′→5′ polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded ‘bubble’, as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each ‘arm’ of the 4-way junction is blunt-ended. Surprisingly, a 3′-tailed duplex, a standard substrate for 3′→5′ helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3′-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom’s or Werner’s syndrome.