Cloning and expression of a functionally active truncated N-glycosylated KSHV ORF4/KCP/kaposica in the methylotrophic yeast Pichia pastoris.
Kaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDS-PAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.
Gomes Pereira
Neuza A
NA
Division of Medical Virology, IIDMM, Faculty of Health Sciences, University of Cape Town, Observatory 7925, Cape Town, South Africa.
Juliano
Maria A
MA
Carmona
Adriana K
AK
Sturrock
Edward D
ED
Kotwal
Girish J
GJ
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D006461:Hemolysis / Q000187:drug effects; D006566:Herpesviridae Infections / Q000517:prevention & control*; D019288:Herpesvirus 8, Human / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics* / Q000494:pharmacology
D017931:DNA Primers; C473771:KSHV complement control protein, Herpesvirus 8; D014764:Viral Proteins