Expression in insect cells and immune reactivity of a 28K tegument protein of human cytomegalovirus

Abstract

The gene encoding the highly antigenic 28K (pp28) tegument phosphoprotein of human cytomegalovirus (HCMV) was expressed in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the recombinant-derived pp28 had mobility on SDS-polyacrylamide gels similar to that of native pp28 from HCMV strain Towne-infected human foreskin fibroblasts (HFFs). In vitro labelling of recombinant Autographa californica nuclear polyhedrosis virusinfected Spodoptera frugiperda cells or of HCMVinfected HFFs with [3Zp]orthophosphate followed by immunoprecipitation showed that both the insect cellderived and HCMV strain Towne-infected fibroblastderived pp28 were phosphorylated. The mobility of pp28 derived from these two sources as well as from extracellular HCMV virions indicated the existence of multiple charged forms of the protein, and a difference in the relative amounts of these forms expressed in HCMV-infected HFFs and recombinant baculovirusinfected insect cells. The recombinant pp28 expressed in insect cells was readily and specifically recognized by antibodies to native pp28, including HCMV-seropositive human serum, and was used in an ELISA to screen human sera for seropositivity.

Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins

“…Of the three putative MCMV matrix proteins, only the M83 product is the target of an easily detectable humoral immune response, as are its putative HCMV counterparts, the HCMV UL82 and UL83 (4). Although antibodies directed against MCMV matrix proteins, like those against HCMV matrix proteins, are likely nonneutralizing and nonprotective, their detection may be useful as a serological marker of the infection (18,61). If the immune response against these matrix proteins can be protective, we predict that it will be so through the induction of cellular responses, specifically through CTLs.…”

Human cytomegalovirus structural proteins

“…In vitro phosphorylation experiments showed that p28 is phosphorylated. The gene coding for ppUL99 has also been expressed in insect cells utilizing a recombinant baculovirus (Giugni et al, 1992). The recombinant ppUL99 was specifically recognized by antibodies to native ppUL99, including HCMV-seropositive human sera.…”

Neutralization of Human Cytomegalovirus by Human CD13-Specific Antibodies

“…An immunoglobulin fraction from pooled serum from CMVseropositive persons (intravenous immune globulin; IVIG) [34] and a CMV glycoprotein B (gB)-specific mouse MAb [25] were used throughout this study as positive controls. IVIG has a high neutralizing titer toward CMV [36] and recognizes a number of proteins in lysates from CMV-infected cells by both immunoprecipitation [36] and immunoblot analysis [37] (including the products of CMV UL99 pp28 [37] and UL83 pp65 genes [data not shown]). The CMV neutralizing activity of serum from seropositive persons is primarily directed against gB [38,39].…”

“…Cellular membrane protein (10 j.tg) and protein from purified enveloped CMV virion and dense body preparations (30 j.tg) were separated by SDS-PAGE on 6.0% gels according to the method of Laemmli [44] and processed for immunoblot. For immunoblot 'analysis, proteins were transferred electrophoretically from the gel to a nitrocellulose membrane (BioRad, Richmond, CA) as described [37]. The immunoreactive bands were detected by incubating the nitrocellulose membrane with a CD I3-specific MAb (3D8; 5 j.tg/mL), the isotype control (UPC 10; 5 j.tg/mL), or the CD 13specific MAb preabsorbed with 10 j.tg/mL of a soluble form of human CD13, followed by a biotinylated goat anti-mouse IgG (Sigma) and a horseradish peroxidase-conjugated streptavidin (Boehringer Mannheim, Indianapolis).…”