Human Cytomegalovirus Virions Research Articles

Characterization of IgG Fc Receptors Induced by Human Cytomegalovirus

Immunoglobulin G Fc receptors (IgG FcRs) induced by human cytomegalovirus (HCMV) were isolated from solubilized HCMV-infected cell extracts by IgG affinity chromatography and analysed by Western blotting using 125I-labelled human IgG and Fc. FcR species of Mr 130K, 65K, 50K and 38K were found to mediate the binding of IgG. The 130K and 65K FcRs were also detected on HCMV virions. All four FcRs were reactive with a murine monoclonal antibody to herpes simplex virus glycoprotein E. Anti-HCMV antibodies markedly inhibited the binding of 125I-labelled human IgG Fc to the 130K and 50K species but inhibited to a lesser extent the binding to the 65K FcR.

Identification of the major capsid protein gene of human cytomegalovirus

The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus. The predicted length of the HCMV MCP was 1,370 amino acids. Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus. A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene. Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases. A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits. The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions. A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots.

Genomic locus for a 140 kDa structural protein (pp150) of human cytomegalovirus in strains Towne and AD169

An HCMV specific clone was isolated from a genomic library of human cytomegalovirus (HCMV) DNA cloned into the expression vector λgt11. This clone (λ111-1) expressed an HCMV/β-galactosidase fusion protein which was reactive with rabbit antibody prepared against purified HCMV virions and dense bodies as well as human HCMV immune serum. By probing Western blots of HCMV virion proteins or HCMV-infected cells with antibody prepared against the fusion protein, the authentic gene product of clone λ111-1 was identified as a high molecular weight polypeptide of 140. Probing the restriction digests of HCMV DNA with insert DNA from the immunoreactive λgt11 clone permitted us to localize the coding sequence for the 140 kDa polypeptide to the long unique region (map coordinates of 0.16–0.18) on HCMV Towne and AD169 genomes.

Genomic localization of the gene encoding a 32-kDa capsid protein of human cytomegalovirus

We have determined the map position of a viral gene encoding a 32-kDa late structural protein of human cytomegalovirus (HCMV) using a murine monoclonal antibody. This monoclonal antibody was reactive with two protein bands of 32 and 27 kDa in HCMV-infected cell lysates and with a single 32-kDa protein band in HCMV virions as detected by immunoblot analysis. When purified HCMV envelope preparation was used for immunoblotting, the monoclonal antibody did not display a detectable band. We used this monoclonal antibody to screen a cDNA library that was constructed from poly(A) + RNA of late HCMV-infected cells and cloned into the expression vector λgt1 1. A cDNA clone that expressed an immunoreactive epitope of the late HCMV protein fused to β-galactosidase was identified. Probing the restriction digests of HCMV (Towne and AD169) DNA with insert DNA from the immunoreactive λgt1 1 clone permitted us to localize the coding sequence within the long unique region between map coordinates of 0.62 and 0.64 of HCMV Towne and AD169 genomes. Using the same probe, a single transcript of 1.4 kb was detected in total RNA from HCMV-infected cells at late times after infection.

Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.

A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine.

Structural and immunological characterization of the intracellular forms of an abundant 68,000 Mr human cytomegalovirus protein.

Murine monoclonal antibodies and polyvalent monospecific antisera reactive with an abundant 68,000 Mr (p68) human cytomegalovirus (CMV) virion protein were used to characterize this protein within CMV-infected cells. The protein was found to partition within the nucleus and cytoplasm of infected cells. Pulse-chase analysis indicated the p68 was degraded into three proteins of 52,000, 51,000 and 50,000 Mr which were found only within infected cells. Both cellular forms as well as the virion p68 were phosphorylated but non-glycosylated. The p68 was synthesized shortly after infection and in the presence of cytosine arabinoside, an inhibitor of viral DNA replication. Studies with monospecific antisera and a panel of monoclonal antibodies specific for the p68 suggested that this protein was not expressed on the surface of infectious virions or infected cells.

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.

Mapping of the major glycoprotein gene of human cytomegalovirus.

The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome. A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector lambda gt11. A cDNA clone was identified which synthesized part of the glycoprotein. It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0.344 and 0.380 of HCMV virion DNA.

Human cytomegalovirus virion-associated protein with kinase activity.

Protein kinase activity was detected in immunoprecipitates of human cytomegalovirus virions and infected cells by using a monoclonal antibody directed against an abundant 68,000-dalton virion structural protein. Purification of this protein by electrophoresis confirmed that the kinase activity was associated with this protein. The kinase activity was dependent on divalent cations (Mg2+, Mn2+) and cyclic nucleotide independent and exhibited optimal activity at pH 7 to 8. The kinase phosphorylated threonine and serine but not tyrosine.

A murine monoclonal antibody recognising a single glycoprotein within a human cytomegalovirus virion envelope glycoprotein complex.

Nonionic detergent solubilised polypeptides from highly purified human cytomegalovirus virions were used as immunogens to produce murine monoclonal antibody secreting hybridomas. One monoclonal antibody was shown, by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), to precipitate three glycoproteins with molecular weights 52, 95, and 130 (all X 10(3)) and one minor component with a molecular weight of 50 X 10(3). When virion envelope components were first separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes, this monoclonal antibody recognised two related components with molecular weights 50 and 52 (both X 10(3)). Immunofluorescence studies suggested that these viral antigens were associated with membrane systems of virus-infected cells and were particularly abundant late in infection.

Characterization of the human cytomegalovirus envelope glycoproteins.

Virions of human cytomegalovirus were shown to contain two discrete membranes. An outer, loose fitting, membrane was sensitive to osmotic shock and could be partially removed by diluting buffered preparations of virions with water. Purified virions were shown, by SDS-polyacrylamide gel electrophoresis of virion membranes labelled by carbohydrate-specific procedures, to contain five glycoproteins with molecular weights of 52, 67, 95, 130 and 250, all X 10(3). Digestion of virions with endoglycosidases revealed that there were structural differences between the carbohydrate portions of the glycoproteins. All five glycoproteins were recognized by antibodies present in pools of human convalescent sera.

Characterization of monoclonal antibodies and polyclonal immune sera directed against human cytomegalovirus virion proteins

Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp58-gp13O, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotein, pp71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, pp155, and (4) a virion-associated phospho-protein, pp29. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 × 10 3 PFU/ml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation.

Defective virions of human cytomegalovirus

Passage of human cytomegalovirus at high multiplicity of infection generated defective virus particles which banded in CsCI at a lower buoyant density than standard virus. The DNA from defective virions banded at a lower buoyant density in CsCl than standard DNA and sedimented in sucrose gradients similar to standard DNA. Contour length measurements of DNA from defective virions revealed various classes of DNAs ranging in molecular weight from 40 to 120 x 10 6; this was in contrast to standard DNA with a molecular weight of 150 x 10 6.