Abduction of Chemokine Elements by Herpesviruses

Chemokines play a key role in orchestrating leukocytic recruitment during inflammatory responses, including those to viral infections. Chemokines are soluble cytokines which mediate their effects through specific G protein-coupled, seven-transmembrane receptors which are expressed on a wide range of cells, including monocytes, T-cells, dendritic cells, and NK cells. Analyses of herpesvirus genomes have revealed that these viral pathogens encode their own versions of both chemokines and chemokine receptors. Viral genes encoding chemokine elements were likely to have been acquired from the host genome and have been remodeled during virus evolution to presumably optimize function or acquire new properties not displayed by their cellular homologues. Virus-encoded chemokines and chemokine receptors are important players in the continuing confrontation between viruses and their mammalian hosts. Detailed characterization of these elements will provide a better understanding of how the immune system responds to viral infection and may suggest new antiviral drug targets and new avenues for the development of antiviral therapies. We will review here the chemokine elements encoded by herpesviruses and how they may aid viral infection and propagation.     

Modulation of cell signaling pathways by kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, has been associated with the development of Kaposi's sarcoma, pleural effusion lymphoma, and multicentric Castleman's disease. KSHV is a double-stranded DNA virus that has been classified as a gammaherpesvirus. The viral genome is approx, 160 kb long and encodes for several genes that are involved in cell signaling pathways. These include genes that are unique to the virus as well as viral homologues of cellular genes. The latter are likely to have been usurped from the host genome and include both virokines and viral receptor proteins. This article reviews how these KSHV proteins modulate cellular signal transduction pathways.     

Human herpesvirus 7 open reading frame U12 encodes a functional beta-chemokine receptor

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3beta (ELC/MIP-3beta) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a beta-chemokine, ELC/MIP-3beta.     

Human herpesvirus 8 encodes a homolog of interleukin-6.

Kaposi's sarcoma is a multifocal lesion that is reported to be greatly influenced by cytokines such as interleukin-6 (IL-6) and oncostatin M. DNA sequences of a novel human gammaherpesvirus, termed human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus, have been identified in all epidemiological forms of Kaposi's sarcoma with high frequency. The presence of HHV-8 DNA is also clearly associated with certain B-cell lymphomas (body cavity-based lymphomas) and multicentric Castleman's disease. Sequence analysis of a 17-kb fragment revealed that adjacent to a block of conserved herpesvirus genes (major DNA-binding protein, glycoprotein B, and DNA polymerase), the genome of HHV-8 encodes structural homolog of IL-6. This cytokine is involved not only in the pathogenesis of Kaposi's sarcoma but also in certain B-cell lymphomas and multicentric Castleman's disease. The viral counterpart of IL-6 (vIL-6) has conserved important features such as cysteine residues involved in disulfide bridging or an amino-terminal signal peptide. Most notably, the region known to be involved in receptor binding is highly conserved in vIL-6. This conservation of essential features and the remarkable overlap between diseases associated with HHV-8 and diseases associated with IL-6 disregulation clearly suggest that vIL-6 is involved in HHV-8 pathogenesis.     

Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4(+) T lymphocytes expressing a nonnaive phenotype, while CD8(+) T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4(+) and CD8(+) T cells, whereas in HHV-6B-infected tissue CD4(+) T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.     

Galpha protein selectivity determinant specified by a viral chemokine receptor-conserved region in the C tail of the human herpesvirus 8 g protein-coupled receptor

The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-kappaB, mitogen-activated protein kinase, and Ca(2+) signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Galpha protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Galpha(i) (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [(35)S]GTPgammaS incorporation assays revealed preferential coupling of vGPCR.15 to Galpha(q) and an inability of vGPCR.8 to couple functionally to Galpha(q). However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Galpha(q). Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Galpha interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Galpha protein coupling specificity.     

HHV8-encoded vMIP-I selectively engages chemokine receptor CCR8. Agonist and antagonist profiles of viral chemokines.

Uncertainty regarding viral chemokine function is mirrored by an incomplete knowledge of host chemokine receptor usage by the virally encoded proteins. One such molecule is vMIP-I, a C-C type chemokine of undefined function and binding specificity, encoded by the Kaposi's sarcoma herpesvirus HHV-8. We report here that vMIP-I binds to and induces cytosolic [Ca(2+)] signals in human T cells selectively through CCR8, a CC chemokine receptor associated with Th2 lymphocytes. Furthermore, using a panel of 65 different human, viral, and rodent chemokines, we have established a comprehensive ligand binding "fingerprint" for CCR8. The receptor exhibits marked "high" affinity (K(d) < 15 nM) only for four chemokines, three of them of viral origin: vMIP-I, vMIP-II, vMCC-I, and human I-309. A previously unreported second class of lower affinity ligands includes MCP-3 and possibly two other viral chemokines. vMIP-I and I-309 appear to act as CCR8 agonists: binding to and inducing cytosolic [Ca(2+)] elevation through the receptor. By contrast, vMIP-II and vMCC-I act as potent antagonists: binding without inducing signaling, and blocking the effects of I-309 and vMIP-I. These results suggest a ligand hierarchy for CCR8, identifying vMIP-I as a selective viral chemokine agonist. CCR8 may thus engage a specific subset of chemokines with the potential to regulate each other during viral infection and immune regulation.     

Unique role of the chemokine domain of fractalkine in cell capture. Kinetics of receptor dissociation correlate with cell adhesion

The chemokine fractalkine (FK) has two structural features that make it unique in the chemokine family: a CX(3)C motif and an extended carboxyl terminus that anchors it to the cell surface. This mucin-like stalk or an equivalent spacer is required for FK to mediate the adhesion of cells expressing its receptor, CX(3)CR1. To determine whether the ability of FK to act as a cell adhesion molecule is due to the unique presentation of a chemokine domain on a stalk or to properties of the chemokine domain itself, we created a series of chimeras in which other soluble chemokines (RANTES (regulated on activation normal T cell expressed), monocyte chemoattractant protein 1, macrophage inflammatory protein 1 beta, secondary lymphoid tissue chemokine, and interleukin 8) were fused to the mucin stalk. When tested in a static-cell adhesion assay, many of these chemokine chimeras demonstrated activity equivalent to that of FK. In flow assays, however, none of the chimeras captured cells as efficiently as FK. Interestingly, FK captured cells expressing either CX(3)CR1 or the viral receptor US28. Cells bound to FK without rolling or detaching, whereas the interleukin 8 and monocyte chemoattractant protein 1 chimeras induced primarily cell rolling and detaching, respectively. In binding studies, FK has a significantly slower off-rate from its receptors than any of the other chemokine chimeras had for their cognate receptors. We conclude that presentation of a chemokine atop a mucin-like stalk is not, in and of itself, sufficient to capture cells. The unique ability of FK to mediate adhesion under flow may be a function of its slow receptor off-rate.     

Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor’s cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8–related diseases.

Herpesviruses are known to hijack cellular receptors to enter cells, but this study shows that human herpesvirus 8 takes this to another level by using its envelope glycoprotein complex gH/gL to mimic the EphA2 receptor’s natural ligands, ephrins.     

Down-regulation of basophil function by human CD200 and human herpesvirus-8 CD200

Human and rodent CD200 are recognized by the inhibitory CD200R, and these molecules play an important role in the regulation of the immune system. Several viruses, such as human herpesvirus-6 (HHV-6), HHV-7, and HHV-8, possess a CD200 homologue, suggesting that these viruses regulate the immune response via CD200R. In this study, we analyzed the effect of human CD200 and the viral CD200 homologues on human CD200R-expressing cells. We found that human CD200R is predominantly expressed on basophils in amounts higher than on other human peripheral blood leukocytes. Furthermore, the viral CD200 homologues as well as human CD200 were recognized by human CD200R, and the activation of basophils was down-regulated by these CD200 proteins. These results suggested that CD200R is an important regulatory molecule of basophil activation. In addition, the presence of CD200 homologues on several viruses suggests a potentially unique relationship between basophil function and viral infection.     

Kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vCCL2 and a highly selective and potent agonist vCCL3

Large DNA viruses such as herpesvirus and poxvirus encode proteins that target and exploit the chemokine system of their host. These proteins have the potential to block or change the orchestrated recruitment of leukocytes to sites of viral infection. The genome of Kaposi sarcoma-associated herpes virus (KSHV) encodes three chemokine-like proteins named vCCL1, vCCL2, and vCCL3. In this study vCCL3 was probed in parallel with vCCL1 and vCCL2 against a panel of the 18 classified human chemokine receptors. In calcium mobilization assays vCCL1 acted as a selective CCR8 agonist, whereas vCCL2 was found to act as a broad spectrum chemokine antagonist of human chemokine receptors, including the lymphotactin receptor. In contrast vCCL3 was found to be a highly selective agonist for the human lymphotactin receptor XCR1. The potency of vCCL3 was found to be 10-fold higher than the endogenous human XCL1 chemokine in respect to phosphatidylinositol turnover and calcium mobilization as well as chemotaxis. High expression of XCR1 was found in placenta and neutrophils by real-time PCR. These data are consistent with reports of different expression profiles for vCCL2 and vCCL3 during the life cycle of KSHV, indicate a novel, sophisticated exploitation by the virus of specifically the lymphotactin receptor by both agonist and antagonist mechanisms, and suggest a unique physiological importance of this (somewhat overlooked) chemokine receptor.     

Human herpesvirus 8 (HHV-8/KSHV) and hematologic malignancies

Human herpesvirus 8 (HHV-8), also defined Kaposi's sarcoma (KS)-associated herpesvirus, was identified by Chang and colleagues in 1994 using purely molecular techniques, before any serological evidence or virus isolation in cell culture could be achieved. HHV-8 is unique among herpesviruses because its prevalence in the general population is low and because it possesses the richest weaponry of viral oncogenes and tumor-promoting factors ever described. Eleven HHV-8-specific genes are homologs of cellular genes, which were hijacked from the host during a long parallel evolution, and at least five of such genes show both in vitro and in vivo transforming ability. HHV-8 is the causative agent of KS, but it has also been associated with different hematologic malignancies, including primary effusion lymphoma (PEL), multicentric Castelman's disease (MCD), MCD-related immunoblastic/plasmablastic lymphoma and various atypical lymphoproliferative disorders. Although low-level silent infection was detected in bone marrow stromal cells from patients with multiple myeloma, a role of HHV-8 in this disease is unlikely. As seen with KS, the incidence of HHV-8-associated lymphoproliferative disorders is increased in the setting of human immunodeficiency virus infection.     

人类8型疱疹病毒的研究进展

人类8型疱疹病毒（human herpesvirus-8,HHV-8）又称卡波氏肉瘤相关疱疹病毒（Kapo- si's sarcoma- associated herpesvirus,KSHV）,是一种新的肿瘤病毒,目前被认为是卡波氏肉瘤（Kaposi's sarcoma,KS）致病因子,并且与primary effusion lymphoma （PEL）和multicentric Castleman's disease（MCD）相关。该病毒编码许多蛋白,包括潜伏感染相关蛋白,裂解感染相关蛋白和HHV-8特有基因表达蛋白,在KS和HHV-8相关疾病的发病中起到关键作用。     

CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.     

Viruses and the TNF-related cytokines,an evolving battle

Tumor necrosis factor (TNF)-related cytokines are critical effector molecules in the immune response to viral pathogens. Engagement of the TNF receptors by their cognate ligands activates apoptotic and non-apoptotic signaling pathways, both of which can mediate antiviral activity. In response, viruses have evolved mechanisms to inhibit signaling by some cytokines of the TNF superfamily. These strategies are largely unique to each class of virus, but are similar in that they all target key regulatory checkpoints of the TNF pathway. In recent years, studies directed towards dissecting the mechanisms of TNF signaling and the viral retort have led to several significant discoveries, and form the basis for this review.