Characterization of Human Herpesvirus-8 K8.1A/B Glycoproteins by Monoclonal Antibodies

Abstract

Human herpesvirus-8 K8.1 gene encodes for two immunogenic class I glycoproteins, K8.1A and B, originating from spliced messages [(1998) Virology 243, 208-217]. The 228-amino-acid-long K8.1A open reading frame (ORF) contains four N-glycosylation sites and the 167-amino-acid-long K8.1B ORF contains three N-glycosylation sites, sharing similar amino- and carboxyl-termini with ORF K8.1A but with an in-frame deletion [(1998) Virology 249, 140-149]. To characterize the K8.1A and B glycoproteins in the infected body cavity-based B cell lymphoma (BCBL-1) cells and in the virion envelopes, monoclonal antibodies (MAbs) recognizing only K8.1A protein or both K8.1A and B proteins were generated. These antibodies reacted with the infected cell membranes and virion envelopes. Stable COS-1 transformant cells expressed the K8.1A and B proteins independently on the plasma membranes. MAbs recognized multiple proteins with molecular weights ranging from 23 to 72 kDa from the BCBL-1 cells and COS-1 cells and the 72 to 68 kDa molecular-weight proteins from the virion particles. The K8.1A is the predominant protein affinity purified from the infected BCBL-1 cells. Digestion with glycosidases show that these proteins contain both N- and O-linked sugars, suggesting that the multiple proteins recognized by the MAbs represent the precursor and product forms of K8.1A and B proteins, and the 72 to 68 kDa molecular-weight proteins represent the virion particle-associated mature forms of these glycoproteins.

Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry

“…The K8.1B cDNA encodes a 167-amino-acid glycoprotein sharing similar amino and carboxy termini with K8.1A but containing an in-frame deletion (10,44). K8.1A is the predominant form detected within infected cells and virion envelopes (62). The K8.1 gene has attracted significant interest due to the fact that it is positionally colinear to the EBV major glycoprotein gp350/220 gene (20), the murine gammaherpesvirus 68 gp150 gene (53), the herpesvirus saimiri ORF51 gene (5), and the bovine herpesvirus 4 BOEFD1 gene (36,48).…”

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

“…We designed an immunotoxin targeting gpK8.1A, based on the previously described murine mAb 4C3. 55 As shown schematically in Figure 1, the variable heavy and light chain cDNAs from the 4C3 hybridoma were isolated and used to generate a sequence encoding the corresponding single chain variable region fragment; this was linked to the cDNA encoding the PE38 moiety of PE, which lacks the normal N-terminal cell binding domain of the native toxin but contains the effector domains involved in translocation and cytotoxicity (ADPribosylation of elongation factor 2). The immunotoxin was expressed in E. coli, the inclusion body fraction was subjected to standard protocols of solubilization, denaturation and refolding, and the protein was purified by sequential ion exchange and size exclusion chromatography 61 (see Materials and Methods).…”

“…Total RNA was extracted from 4C3 hybridoma cells 55 by using the Qiagen RNeasy mini kit. Briefly, 5 x 10 6 cells were disrupted in 350 μl of buffer RLT containing β-mercaptoethanol and homogenized by loading onto a QIAshredder spin column and spinning for 2 min at a maximum speed in a micro centrifuge.…”

“…51 gpK8.1A is a 228 amino acid late lytic glycoprotein [52][53][54] displayed on KSHV virions; 55,56 it binds to heparin sulfate on the target cell surface, 57,58 thereby facilitating virus entry and consequent spread of infection. Antibodies against gpK8.1A are important serological markers for KSHV disease.…”

“…[53][54][55]60 This viral glycoprotein therefore appears to be a potential target for an immunotoxin designed to kill lytically infected cells.…”