scholarly journals Disintegrin-like domain of glycoprotein B regulates Kaposi’s sarcoma-associated herpesvirus infection of cells

2014 ◽  
Vol 95 (8) ◽  
pp. 1770-1782 ◽  
Author(s):  
Lia R. Walker ◽  
Hosni A. M. Hussein ◽  
Shaw M. Akula
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Glycoprotein B ◽  
Binding Motif ◽  
Wild Type ◽  
Infection Rates ◽  
Herpesvirus Infection ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein’s interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.

scholarly journals Integrin αVβ3 Binds to the RGD Motif of Glycoprotein B of Kaposi's Sarcoma-Associated Herpesvirus and Functions as an RGD-Dependent Entry Receptor

2007 ◽  
Vol 82 (3) ◽  
pp. 1570-1580 ◽  
Author(s):  
H. Jacques Garrigues ◽  
Yelena E. Rubinchikova ◽  
C. Michael DiPersio ◽  
Timothy M. Rose
Keyword(s):  
Cho Cells ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Glycoprotein B ◽  
Target Cells ◽  
Kshv Infection ◽  
Rgd Motif ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Blocking Antibodies

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin α3β1 during virus entry. Although the ligand specificity of α3β1 has been controversial, current literature indicates that α3β1 ligand recognition is independent of RGD. We compared α3β1 to the RGD-binding integrin, αVβ3, for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that β3-CHO cells overexpressing αVβ3 specifically bound gB(RGD), whereas α3-CHO cells overexpressing α3β1 did not. Function-blocking antibodies to αVβ3 inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to α3β1 did not. Using affinity-purified integrins and confocal microscopy, αVβ3 bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific αVβ3 antagonists, including cyclic and dicyclic RGD peptides and αVβ3 function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from α3-null mice lacking α3β1 were fully competent for infection by KSHV, and reconstitution of α3β1 function by transfection with α3 cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of α3β1 on the cell surface expression of αVβ3 and on αVβ3-mediated adhesion of α3-CHO cells overexpressing α3β1 were detected, consistent with previous reports of transdominant inhibition of αVβ3 function by α3β1. These observations may explain previous reports of an inhibition of KSHV infection by soluble α3β1. Our studies demonstrate that αVβ3 is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).

scholarly journals Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Xiaojuan Li ◽  
Fanxiu Zhu
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Kaposi's Sarcoma ◽  
Nuclear Export Signal ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

scholarly journals Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jennifer T. Miller ◽  
Haiyan Zhao ◽  
Takashi Masaoka ◽  
Brittany Varnado ◽  
Elena M. Cornejo Castro ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Active Site ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclease Activity ◽  
Divalent Metal ◽  
Wild Type ◽  
Nuclease Domain ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to theHerpesviridaefamily, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

Sign in / Sign up

Export Citation Format

Share Document