Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

Assembly of infectious Kaposi's sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer.

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its ß-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.

Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS and which is endemic in sub-Saharan Africa. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses. Capsid assembly and genome packaging of herpesviruses are prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids-comprising nearly 3,000 proteins and over 1,300 Å in diameter-present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP-MCP interaction-including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain-define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid.

With just one eighth the size of the major capsid protein (MCP), the smallest capsid protein (SCP) of human tumor herpesviruses--Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV)--is vital to capsid assembly, yet its mechanism of action is unknown. Here, by cryoEM of KSHV at 6-Å resolution, we show that SCP forms a crown on each hexon and uses a kinked helix to cross-link neighboring MCP subunits. SCP-null mutation decreased viral titer by 1,000 times and impaired but did not fully abolish capsid assembly, indicating an important but nonessential role of SCP. By truncating the C-terminal half of SCP and performing cryoEM reconstruction, we demonstrate that SCP's N-terminal half is responsible for the observed structure and function whereas the C-terminal half is flexible and dispensable. Serial truncations further highlight the critical importance of the N-terminal 10 aa, and cryoEM reconstruction of the one with six residues truncated localizes the N terminus of SCP in the cryoEM density map and enables us to construct a pseudoatomic model of SCP. Fitting of this SCP model and a homology model for the MCP upper domain into the cryoEM map reveals that SCP binds MCP largely via hydrophobic interactions and the kinked helix of SCP bridges over neighboring MCPs to form noncovalent cross-links. These data support a mechanistic model that tumor herpesvirus SCP reinforces the capsid for genome packaging, thus acting as a cementing protein similar to those found in many bacteriophages.

Organization of capsid-associated tegument components in Kaposi's sarcoma-associated herpesvirus.

UNLABELLED: Capsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a ß-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein. IMPORTANCE: Several capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

KSHV targets multiple leukocyte lineages during long-term productive infection in NOD/SCID mice.

To develop an animal model of Kaposi sarcoma-associated herpesvirus (KSHV) infection uniquely suited to evaluate longitudinal patterns of viral gene expression, cell tropism, and immune responses, we injected NOD/SCID mice intravenously with purified virus and measured latent and lytic viral transcripts in distal organs over the subsequent 4 months. We observed sequential escalation of first latent and then lytic KSHV gene expression coupled with electron micrographic evidence of virion production within the murine spleen. Using novel technology that integrates flow cytometry with immunofluorescence microscopy, we found that the virus establishes infection in murine B cells, macrophages, NK cells, and, to a lesser extent, dendritic cells. To investigate the potential for human KSHV-specific immune responses within this immunocompromised host, we implanted NOD/SCID mice with functional human hematopoietic tissue grafts (NOD/SCID-hu mice) and observed that a subset of animals produced human KSHV-specific antibodies. Furthermore, treatment of these chimeric mice with ganciclovir at the time of inoculation led to prolonged but reversible suppression of KSHV DNA and RNA levels, suggesting that KSHV can establish latent infection in vivo despite ongoing suppression of lytic replication.

[Herpesvirus type 8 and its implication in human pathology].

Herpesvirus type 8 or Kaposi's sarcoma-associated virus has been recently discovered and it is an etiologic agent of several known diseases. It has common features that link it with other representatives of the family Herpesviridae: similar structural elements and genomic organization, and the common mechanisms of replication. Nevertheless, this virus has a number of unique features that make it an interesting matter for investigations and currently central in modern medicine and biology. This overview is to draw attention to this representative of herpesviruses and to outline some epidemiological, pathogenetic, and molecular aspects of this problem.

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the gamma-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of "B-capsid-like" particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.

The molecular pathology of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the eighth and most recently identified human herpesvirus (HHV-8). KSHV was discovered in 1994 by Chang et al. who used representational difference analysis to search for DNA sequences present in AIDS-associated KS but not in adjacent normal skin [1]. The virus has since been shown to be specifically associated with all forms of this disease and has fulfilled all of Hill's criteria for causation (reviewed in ). KSHV is also found in all cases of primary effusion lymphoma and in a plasmablastic variant of multicentric Castleman's disease. Over the last few years a wealth of data has been gained on the role of KSHV genes during infection. This review is an attempt to assemble this information into a more complete picture of how KSHV may cause disease.

Visualization of human herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy.

BACKGROUND: Human herpesvirus type 8 (HHV-8) has been associated with Kaposi's sarcoma, body cavity-based lymphoma (BCBL), and multicentric Castleman's disease through DNA, in situ hybridization, and serologic studies. HHV-8 has been visualized only in HHV-8-positive/Epstein-Barr virus (EBV)-negative/ cytomegalovirus (CMV)-negative BCBL cell lines, but not in HHV-8-positive/EBV-negative/ CMV-negative Kaposi's sarcoma lesions. DESIGN: Kaposi's sarcoma of the skin, lymph node, and spleen from three patients with AIDS were analysed for HHV-8, EBV and CMV DNA by polymerase chain reaction (PCR), for HHV-8 RNA (Tl.1 riboprobe) by in situ hybridization (ISH), for viral inclusions by light microscopy, and for herpesviruses by transmission electron microscopy (TEM). Sections were also labeled with Tl.1 counterstained with CD34, an endothelial cell marker. RESULTS: The skin lesion was DNA PCR-positive for HHV-8 and CMV (nested, but not single PCR), the lymph node was positive for HHV-8 and EBV, and the spleen was positive for only HHV-8. TEM revealed infection by a virus displaying the typical morphology and cytopathicity of herpesviruses. Hexagonal nucleocapsids and mature enveloped virions were present in vasoformative spindle cells and mononuclear cells, often resembling lymphocytes. Extrapolating from TEM to standard light microscopy on hematoxylin and eosin-stained paraffin sections, eosinophilic, targetoid intranuclear inclusions were identified within spindle cells which often lined vascular lumina. The Tl.1-riboprobe labeled CD34+ spindle cells containing intranuclear inclusions, as well as mononuclear cells within Kaposi's sarcoma and residual lymphoid tissue. CONCLUSION: The herpesvirus visualized in Kaposi's sarcoma lesions has morphologic and cytopathic features typical of human herpesviruses, productively infects vasoformative spindle cells and mononuclear cells, and is consistent with HHV-8. It can also form intranuclear inclusions that are identifiable by light microscopy in hematoxylin and eosin sections and by ISH.

Kaposi's sarcoma: breeding ground of herpesviridae - A tour de force over viral evolution (review).

After reviewing the molecular biological basis of prominent theories for the integration of viruses into the earliest forms of living matter, an account is given on the immunoevasive strategies viruses have had to acquire in order to secure their existence against the most sophisticated anti-viral defensive mechanisms evolving in their hosts. Herpes-viridae and Kaposi's sarcoma illustrate the complexity of host-virus relationship. In following the evolutionary steps of simians and hominoids to Homo, it becomes evident that: a) Epstein-Barr virus evolved in Africa and its ancestral viruses are present in cercopithecines and hominoids; b) human herpes-virus-8-related viruses are present in macaques, in S. American primates and in Homo but such isolates from the great apes are missing. Thus interspecies transfer occurred from lower monkeys to Homo but when and at what geographical location? The human retrolentiviruses also jumped species barriers: this occurred recently in Africa, from great apes (chimpanzee and bonobo) to Homo sapiens (except when HIV-2 was transferred to mankind from sooty mangabeys). The matter is further complicated by the long coevolutionary cooperative interactions between herpes- and retrolentiviruses. Of pathological entities suspected to be etiologically affected by such complex viral cooperation, the origin of Reed-Sternberg cells of Hodgkin's disease is singled out for critical analysis. In this article the senior author summarizes his own 52 years of studentship in virology.

Demonstration of human herpesvirus 8 in a case of primary vaginal epithelioid angiosarcoma by in situ hybridization, electron microscopy, and polymerase chain reaction.

We demonstrate the presence of human herpesvirsus 8 (HHV-8) in a primary vaginal location of angiosarcoma (AS) by polymerase chain reaction (PCR), in situ hybridization, and ultrastructural direct visualization of viral particles. The latter two techniques for the first time confirm HHV-8 detection in an AS by PCR; these results contribute to the debate caused by the controversial data produced by the almost exclusive use of PCR for investigating the possible presence of HHV-8 in AS, and its possible implications. Moreover, the investigated AS is the seventh published primary vaginal one, and the fourth unrelated to radiotherapy. Interestingly, the affected patient had used a ring pessary for 10 years because of an uterovaginal prolapse.

Purification of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) and analyses of the structural proteins.

The Kaposis's sarcoma-associated herpesvirus (KSHV) infected BCBL-1 cell line, adapted to and grown in medium containing 10% horse serum, was induced to lytic replication with 12-O-tetradecanoylphorbol-13-acetate (TPA) for virus production. Supernatants from induced cells were filtered through a 0.45-microm filter and virions were concentrated by polyethylene glycol extraction and high speed centrifugation. The virus was purified by a glycerol gradient zonal centrifugation step followed by isopycnic separation using positive density negative viscosity gradients. Two visible bands were detected after the final centrifugation step: an upper band that contained a homogenous population of purified virions and a lower band that contained aggregates of purified virus and other cellular debris. Fractionation of purified virion preparations by SDS-PAGE revealed 32 bands with estimated molecular weights between 19 and 280 K in silver stained gels. The glycoprotein bands in purified virus were identified with biotinylated lectins and horseradish peroxidase-labeled streptavidin. Two lectins were used to identify the KSHV glycoproteins: concanavalin A and Ricinus communis agglutinin I. Eight distinct glycoproteins were detected with these lectins. In addition, antisera from KS patients were used to detect immunoreactive proteins in purified virions. An apparent immunodominant band of Mr 94,000 (94 K) was recognized by patients' antisera. Other proteins detected with some of the KS antisera tested corresponded to molecular weights of 57 K, 70 K, 180 K, 200 K and 240 K. The 94 K band was identified as gp94 by Endo F digestion.

Human herpesvirus 8 in primary effusion lymphoma in an HIV-seronegative male. A case report.

BACKGROUND: AIDS-related body cavity-based lymphoma, or primary effusion lymphoma (PEL), is a distinct clinicopathologic entity that occurs predominantly in immunosuppressed patients infected with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. Although it rarely occurs in human immunodeficiency virus (HIV)-negative patients, we report such a case here. CASE: A 74-year-old male, who was HIV and Epstein-Barr virus (EBV) negative, was admitted to the hospital with dyspnea and chest pain. Chest radiography and computed tomography showed right pleural effusion. Cytologic analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm. Polymerase chain reaction performed on the pleural effusion was positive for HHV-8 and negative for EBV. On molecular studies, the immunoglobulin heavy and kappa light chains were rearranged. Flow cytometry revealed a hyperploid fraction with DNA index of 1.29 expressing CD30. Immunostaining for HHV-8 from a cell block was positive. Electron microscopy revealed lymphomalike cells, many in various stages of apoptosis, with large nucleoli and clusters of viruslike particles in the nucleoplasm. CONCLUSION: A firm diagnosis of PEL can be established by the examination of cells from the lymphomatous effusion by a combination of cytology, molecular genetics, phenotypic features, immunostaining and electron microscopy. To our knowledge, this is the first case in which immunostaining for anti-HHV-8 monoclonal antibodies was used to support the diagnosis.

Direct visualization of the putative portal in the Kaposi's sarcoma-associated herpesvirus capsid by cryoelectron tomography.

Genetic and biochemical studies have suggested the existence of a bacteriophage-like, DNA-packaging/ejecting portal complex in herpesviruses capsids, but its arrangement remained unknown. Here, we report the first visualization of a unique vertex in the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid by cryoelectron tomography, thus providing direct structural evidence for the existence of a portal complex in a gammaherpesvirus. This putative KSHV portal is an internally localized, umbilicated structure and lacks all of the external machineries characteristic of portals in DNA bacteriophages.

Changes occurring on the cell surface during KSHV reactivation.

Following an infection, Kaposi's sarcoma-associated herpes virus (KSHV) exists predominantly in its latent state, with only 1-2% of infected cells undergoing lytic reactivation. We have previously demonstrated along with others a relationship between lytic reactivation and cell cycle progression (Bryan et al., 2006. J. Gen. Virol. 87: 519; McAllister et al., 2005. J. Virol. 79: 2626). Infected cells in the S phase are much more likely to undergo lytic reactivation when compared to those in G(0)/G(1) phase. Through the use of scanning electron microscopy (SEM), we analyzed changes occurring on the surface of cells undergoing KSHV reactivation. KSHV reactivation was observed predominantly in cells with smoother surface topology; a hallmark of cells derived from S phase. Interestingly, during the late stages of the reactivation process, we observed KSHV particles to egress cells through budding. Taken together, based on scanning electron microscopy and transmission electron microscopy evidences, we demonstrate for the first time the existence of a direct link between cell surface topology, cell cycle progression and KSHV reactivation.

[Human herpesvirus 8]. / L'herpesvirus humain 8.

Human herpesvirus 8 (HHV-8, KSHV) is a novel virus for which fragments of genomic sequence were identified in 1994. This was done by means of a differential amplification technique applied to the DNA of Kaposi's sarcoma lesions and normal tissues obtained from the same individual. The analysis of nucleotide sequence has shown that HHV-8 is closely related to herpesvirus saimiri and Epstein-Barr virus, two members of Gammaherpesvirinae sub-family. The virus has been observed by means of electron microscopy in chronically infected cell lines. Serological studies are still preliminary but seem to demonstrate a low prevalence of HHV-8 infection among the general population at least in Western countries, HHV-8 is mainly detected by means of polymerase chain reaction (PCR) and molecular hybridization. HHV-8 detection in human tissues is strongly associated with three diseases: Kaposi's sarcoma, body-cavity-based lymphomas and Castleman's disease. The demonstration of the causative role of HHV-8 in the occurrence of these three diseases, particularly Kaposi's sarcoma, would have major consequences for their diagnosis and treatment.

Ultrastructural characterization of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) in Kaposi's sarcoma lesions: electron microscopy permits distinction from cytomegalovirus (CMV).

Kaposi's sarcoma (KS) has been shown by molecular techniques to be associated with infection with human herpesvirus 8 (HHV8/KSHV), but specific ultrastructural characterization of the virus has been impaired by the frequent presence in these lesions of other herpesviruses, particularly cytomegalovirus (CMV). Since the ultrastructural appearance of HHV8/KSHV has been studied in the cell line KS-1 uninfected with other viruses including CMV, it was possible to undertake a comparative study of CMV and HHV8/KSHV in KS lesions. HHV8/KSHV was sparsely present and lytic infection was restricted to endothelial cells. The following specific ultrastructural features allowed distinction between HHV8/KSHV and CMV: the viral particles were more delicate and less numerous in cases of HHV8/KSHV infection; the viral tegument was more electron-dense in CMV than in HHV8/KSHV; dense bodies characteristic of CMV were absent in HHV/KSHV; complete CMV viral particles were more variable in size and generally larger (150-200 nm) than HHV8/KSHV (120-150 nm); and finally, the viral envelope was more pleomorphic in CMV than in KSHV/HHV8. Similarities between CMV and HHV8/KSHV included the basic structure of the nucleocapsids and the presence of capsids lacking central DNA cores (so-called non-infectious enveloped particles). These observations show that electron microscopy can be used to identify HHV8/KSHV and confirm the relationship between HHV8/KSHV and KS.

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus.

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.