scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus

2005 ◽  
Vol 79 (2) ◽  
pp. 1191-1206 ◽  
Author(s):  
Pramod P. Naranatt ◽  
Harinivas H. Krishnan ◽  
Marilyn S. Smith ◽  
Bala Chandran
Keyword(s):  
Cell Surface ◽  
Cell Signaling ◽  
Host Cell ◽  
Rho Gtpases ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Viral Dna ◽  
Host Cell Signaling

ABSTRACT Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the α3β1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.

scholarly journals Cellular Receptors Involved in KSHV Infection

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 118
Author(s):  
Emma van der Meulen ◽  
Meg Anderton ◽  
Melissa J. Blumenthal ◽  
Georgia Schäfer
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cell Types ◽  
Target Cells ◽  
Specific Cell ◽  
Cellular Receptors ◽  
Diverse Range ◽  
Kshv Infection

The process of Kaposi’s Sarcoma Herpes Virus’ (KSHV) entry into target cells is complex and engages several viral glycoproteins which bind to a large range of host cell surface molecules. Receptors for KSHV include heparan sulphate proteoglycans (HSPGs), several integrins and Eph receptors, cystine/glutamate antiporter (xCT) and Dendritic Cell-Specific Intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This diverse range of potential binding and entry sites allows KSHV to have a broad cell tropism, and entry into specific cells is dependent on the available receptor repertoire. Several molecules involved in KSHV entry have been well characterized, particularly those postulated to be associated with KSHV-associated pathologies such as Kaposi’s Sarcoma (KS). In this review, KSHV infection of specific cell types pertinent to its pathogenesis will be comprehensively summarized with a focus on the specific cell surface binding and entry receptors KSHV exploits to gain access to a variety of cell types. Gaps in the current literature regarding understanding interactions between KSHV glycoproteins and cellular receptors in virus infection are identified which will lead to the development of virus infection intervention strategies.

scholarly journals Early Events in Kaposi's Sarcoma-Associated Herpesvirus Infection of Target Cells

2009 ◽  
Vol 84 (5) ◽  
pp. 2188-2199 ◽  
Author(s):  
Bala Chandran
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Mode Of Entry ◽  
Successful Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Cell Signaling Pathways

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently identified member of the herpesvirus family, infects a variety of target cells in vitro and in vivo. This minireview surveys current information on the early events of KSHV infection, including virus-receptor interactions, involved envelope glycoproteins, mode of entry, intracellular trafficking, and initial viral and host gene expression programs. We describe data supporting the hypothesis that KSHV manipulates preexisting host cell signaling pathways to allow successful infection. The various signaling events triggered by infection, and their potential roles in the different stages of infection and disease pathogenesis, are summarized.

scholarly journals Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells

2006 ◽  
Vol 80 (3) ◽  
pp. 1167-1180 ◽  
Author(s):  
Harinivas H. Krishnan ◽  
Neelam Sharma-Walia ◽  
Daniel N. Streblow ◽  
Pramod P. Naranatt ◽  
Bala Chandran
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Viral Entry ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Viral Dna ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.

scholarly journals Human Herpesvirus 8 Envelope Glycoprotein B Mediates Cell Adhesion via Its RGD Sequence

2003 ◽  
Vol 77 (5) ◽  
pp. 3131-3147 ◽  
Author(s):  
Fu-Zhang Wang ◽  
Shaw M. Akula ◽  
Neelam Sharma-Walia ◽  
Ling Zeng ◽  
Bala Chandran
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Focal Adhesion ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Target Cells ◽  
Herpesvirus 8 ◽  
Rgd Sequence

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes α3β1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM) and a gBΔTM mutant (gBΔTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBΔTM, gBΔTM-RGA, and ΔTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBΔTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBΔTM-RGA and ΔTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBΔTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBΔTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBΔTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBΔTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBΔTM protein to the target cells but not by gBΔTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBΔTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.

scholarly journals Kaposi’s sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging

2018 ◽  
Author(s):  
Matthew R Gardner ◽  
Britt A Glaunsinger
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Dna Packaging ◽  
Viral Dna ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Genome Encapsidation

AbstractHerpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However,in vitrobiochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.ImportanceKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double stranded DNAin vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Matthew R. Gardner ◽  
Britt A. Glaunsinger
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Dna Packaging ◽  
Viral Dna ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Genome Encapsidation

ABSTRACTHerpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However,in vitrobiochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double-stranded DNAin vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Tenaya K. Vallery ◽  
Johanna B. Withers ◽  
Joana A. Andoh ◽  
Joan A. Steitz
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.IMPORTANCELate in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

scholarly journals The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity

2000 ◽  
Vol 74 (24) ◽  
pp. 11977-11982 ◽  
Author(s):  
Junsoo Park ◽  
Taegun Seo ◽  
Seungmin Hwang ◽  
Daeyoup Lee ◽  
Yousang Gwack ◽  
...  
Keyword(s):  
Host Cell ◽  
Kaposi's Sarcoma ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Kaposi’S Sarcoma ◽  
Cellular Transcription Factor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma. KSHV encodes K-bZIP (open reading frame K8), a protein that belongs to the basic region-leucine zipper (bZIP) family of transcription factors. Here we show that K-bZIP associates with the cellular transcription factor p53 directly in vitro and in vivo. This interaction requires the bZIP domain of K-bZIP and the carboxy-terminal region (amino acids 300 to 393) of p53. We also show that K-bZIP represses the transcriptional activity of p53 which is required for apoptosis of the host cell. These results imply that K-bZIP blocks p53-mediated host cell death through its interaction with p53.

scholarly journals Envelope Glycoprotein gB of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Egress from Infected Cells

2005 ◽  
Vol 79 (17) ◽  
pp. 10952-10967 ◽  
Author(s):  
Harinivas H. Krishnan ◽  
Neelam Sharma-Walia ◽  
Ling Zeng ◽  
Shou-Jiang Gao ◽  
Bala Chandran
Keyword(s):  
Envelope Glycoprotein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Signal Pathways ◽  
Virus Binding ◽  
Viral Dna ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) envelope glycoprotein gB interacts with cell surface heparan sulfate (HS) and α3β1 integrin and plays roles in the initial binding and entry into the target cells and in the induction of preexisting host cell signal pathways. To define gB function further, using a bacterial artificial chromosome (BAC) system carrying the KSHV genome (BAC36wt-KSHV), we constructed a recombinant virus genome with the gB open reading frame (ORF) deleted by replacing a 2-kb gB ORF with a 1.3-kb Kanr gene. Stable 293T cells carrying BAC36wt-KSHV and ΔgBBAC36-KSHV genomes were generated. Transcript analyses and immunoprecipitation reactions confirmed the absence of gB in the 293T-ΔgBBAC36 cells. When monolayers of 293T-BAC36wt and 293T-ΔgBBAC36 cells were induced with tetradecanoylphorbol-13-acetate, infectious virus was detected only from the 293T-BAC36wt cell supernatants. No significant amount of DNase I-resistant viral DNA was detected in the supernatants of 293T-ΔgBBAC36 cells. BAC36wt-KSHV infected the target cells, and in contrast, no viral DNA and transcripts could be detected in cells infected with ΔgBBAC36-KSHV. Electron microscopy of 293T-ΔgBBAC36 cells revealed capsids in the nuclei, cytoplasmic vesicles with core-containing capsids, and occasional enveloped virions in the cytoplasm. However, enveloped virus particles were observed in the extracellular compartments of 293T-BAC36wt cells only and not in 293T-ΔgBBAC36 cells. Transfection of 293T-ΔgBBAC36 cells with plasmid expressing full-length gB restored the recovery of infectious KSHV in the supernatant. These results suggest that, besides its role in virus binding and entry into the target cells, KSHV gB also plays a role in the maturation and egress of virus from the infected cells.

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