scholarly journals The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

2006 ◽  
Vol 43 (10) ◽  
pp. 1665-1675 ◽  
Author(s):  
Linda Mark ◽  
Wen H. Lee ◽  
O. Brad Spiller ◽  
Bruno O. Villoutreix ◽  
Anna M. Blom
Keyword(s):  
Amino Acids ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cell Surfaces ◽  
Complement Control Protein ◽  
Charged Amino Acids ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Control Protein ◽  
Positively Charged

scholarly journals Dissecting the Regions of Virion-Associated Kaposi's Sarcoma-Associated Herpesvirus Complement Control Protein Required for Complement Regulation and Cell Binding

2006 ◽  
Vol 80 (8) ◽  
pp. 4068-4078 ◽  
Author(s):  
O. B. Spiller ◽  
L. Mark ◽  
C. E. Blue ◽  
D. G. Proctor ◽  
J. A. Aitken ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Heparin Binding ◽  
Cell Binding ◽  
Complement Control Protein ◽  
Factor I ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Control Protein

ABSTRACT Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Is a Coregulator of K-Rta: Physical Association and Promoter-Dependent Transcriptional Repression

2003 ◽  
Vol 77 (2) ◽  
pp. 1441-1451 ◽  
Author(s):  
Yoshihiro Izumiya ◽  
Su-Fang Lin ◽  
Thomas Ellison ◽  
Ling-Yu Chen ◽  
Chie Izumiya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Dependent Manner ◽  
Basic Leucine Zipper ◽  
Interaction Domain ◽  
Ample Evidence ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

scholarly journals A Domain in the C-Terminal Region of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Affects Transcriptional Activation and Binding to Nuclear Heterochromatin

2003 ◽  
Vol 77 (12) ◽  
pp. 7093-7100 ◽  
Author(s):  
Abel Viejo-Borbolla ◽  
Emrah Kati ◽  
Julie A. Sheldon ◽  
Kavita Nathan ◽  
Karin Mattsson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Matrix ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Terminal Region ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Carboxy Terminal ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Episomal Dna

ABSTRACT The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.

scholarly journals The Cytoplasmic Terminus of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein B Is Not Essential for Virion Egress and Infectivity

2008 ◽  
Vol 82 (14) ◽  
pp. 7144-7154 ◽  
Author(s):  
R. Subramanian ◽  
O. D'Auvergne ◽  
Haixia Kong ◽  
K. G. Kousoulas
Keyword(s):  
Amino Acids ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cytoplasmic Domain ◽  
Glycoprotein B ◽  
Virus Infectivity ◽  
Mrna Levels ◽  
293 Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted α-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.

scholarly journals A Mobile Functional Region of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein Independently Regulates DNA Binding and Protein Abundance

2008 ◽  
Vol 82 (19) ◽  
pp. 9700-9716 ◽  
Author(s):  
Pey-Jium Chang ◽  
Duane Shedd ◽  
George Miller
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Kaposi's Sarcoma ◽  
Regulatory Region ◽  
Kaposi’S Sarcoma ◽  
Protein Abundance ◽  
Transactivation Domain ◽  
Functional Region ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The protein encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) functions as a transcriptional activator and in lytic viral DNA replication to mediate the switch from latent viral infection to the lytic phase. Here we identify regulatory regions of ORF50 protein that independently control DNA binding and abundance of the protein. One region contains a DNA-binding inhibitory sequence (DBIS) located between amino acids (aa) 490 and 535 of ORF50. A cluster of basic amino acids in this sequence is important in inhibiting DNA binding. The DBIS can function at the N or C terminus or internally in the ORF50 protein. Since the DBIS is functional in ORF50 protein purified from Escherichia coli, it is likely to work through an intramolecular mechanism. The second regulatory region, a protein abundance regulatory signal (PARS), consists of two components. Component I of the PARS overlaps the DBIS but can be differentiated from the DBIS by specific substitution of basic amino acid residues. Component II of PARS is located between aa 590 and 650. Mutation or deletion of either component results in abundant expression of ORF50 protein. When the two-component PARS was fused to a heterologous protein, Glutathione S-transferase, the fusion protein was unstable. Mutations in the DBIS or PARS impair the capacity of ORF50 to activate direct and indirect target viral promoters. Since these overlapping regulatory motifs are located in the C-terminal transactivation domain, they are likely to be important in controlling many actions of ORF50 protein.

scholarly journals Transcriptional Activation by the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Is Facilitated by an N-Terminal Chromatin-Binding Motif

2004 ◽  
Vol 78 (18) ◽  
pp. 10074-10085 ◽  
Author(s):  
Lai-Yee Wong ◽  
Gerald A. Matchett ◽  
Angus C. Wilson
Keyword(s):  
Amino Acids ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Host Cells ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Chromatin Binding ◽  
C Terminus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT In immunocompromised patients, infection with Kaposi's sarcoma-associated herpesvirus (KSHV) can give rise to Kaposi's sarcoma and several lymphoproliferative disorders. In these tumors, KSHV establishes a latent infection in many of the rapidly proliferating and morphologically abnormal cells. Only a few viral gene products are expressed by the latent virus, and one of the best characterized is the latency-associated nuclear antigen (LANA), a nuclear protein required for the maintenance of viral episomal DNA in the dividing host cell. LANA can also activate or repress an assortment of cellular and viral promoters and may contribute to pathogenesis by allowing the proliferation and survival of host cells. Here we show that activation of the human E2F1 and cyclin-dependent kinase-2 (CDK2) promoters requires elements from both the N- and C-terminal regions of LANA. Deletion of the first 22 amino acids, which are necessary for episome tethering, does not affect nuclear localization but significantly reduces transactivation. Within the deleted peptide, we have identified a short sequence, termed the chromatin-binding motif (CBM), that binds tightly to interphase and mitotic chromatin. A second chromatin-binding activity resides in the C terminus but is not sufficient for optimal transactivation. Alanine substitutions within the CBM reveal a close correlation between the transactivation and chromatin binding activities, implying a mechanistic link. In contrast to promoter activation, we find that the 223 amino acids of the LANA C terminus are sufficient to inhibit p53-mediated activation of the human BAX promoter, indicating that the CBM is not required for all transcription-related functions.

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