ABSTRACT
Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.
Subject(s)
Chemokines, CC/metabolism , Herpesviridae Infections/immunology , Immunity, Innate , Macrophages/immunology , Muromegalovirus/physiology , Viral Envelope Proteins/metabolism , Viral Proteins/metabolism , Virus Internalization , Animals , Cell Line , Cells, Cultured , Chemokines, CC/chemistry , Chemokines, CC/genetics , Female , Herpesviridae Infections/metabolism , Herpesviridae Infections/pathology , Herpesviridae Infections/virology , Liver/immunology , Liver/pathology , Liver/virology , Macrophages/pathology , Macrophages/virology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/pathology , Macrophages, Peritoneal/virology , Mice , Mice, Inbred BALB C , Muromegalovirus/immunology , Mutation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Specific Pathogen-Free Organisms , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Virion/immunology , Virion/physiologyABSTRACT
Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.
Subject(s)
Capsid Proteins/genetics , Muromegalovirus/genetics , Nuclear Envelope/virology , Nuclear Proteins/genetics , Virus Replication/genetics , Alleles , Animals , Blotting, Southern , Blotting, Western , Capsid Proteins/metabolism , Cell Line , Enzyme-Linked Immunosorbent Assay , Genetic Complementation Test , Immunoprecipitation , Mice , Microscopy, Confocal , Microscopy, Electron, Transmission , Muromegalovirus/growth & development , Mutation/genetics , Nuclear Proteins/metabolism , Plasmids/genetics , Polymerase Chain ReactionABSTRACT
Certain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a ß-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split ß-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC-PCA, we tested peptide inhibitors of the pUL50-pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC-PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50-pUL53 interaction assay allows a simple cell free test for small molecular inhibitors.