ABSTRACT
Pneumonia virus of mice (PVM; family Paramyxoviridae) is a natural pathogen of rodents that reproduces important clinical features of severe respiratory syncytial virus infection in humans. As anticipated, PVM infection induces transcription of IFN antiviral response genes preferentially in wild-type over IFN-alphabetaR gene-deleted (IFN-alphabetaR-/-) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-gamma-inducible protein-10, and TLR-3 in lung tissue of IFN-alphabetaR-/- mice when compared with wild type. No differential expression of chemokines MIP-1alpha or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-alphabetaR-/- mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-alphabetaR-/- mice (50% survival at 10.8 +/- 0.6 days vs the wild type at 9.0 +/- 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-alphabetaR-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo.
Subject(s)
Gene Deletion , Lung/pathology , Pneumovirus Infections/immunology , Pneumovirus/immunology , Receptors, Interferon/deficiency , Receptors, Interferon/genetics , Animals , Chemokine CCL3 , Chemokine CCL4 , Chemokine CXCL10 , Chemokine CXCL2 , Chemokines/biosynthesis , Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/virology , Interferon Type I/biosynthesis , Interferon Type I/genetics , Leukocytes/pathology , Lung/immunology , Lung/virology , Macrophage Inflammatory Proteins/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocyte Chemoattractant Proteins/biosynthesis , Monocyte Chemoattractant Proteins/genetics , Pneumovirus/physiology , Pneumovirus Infections/mortality , Pneumovirus Infections/pathology , RNA, Messenger/metabolism , Virus Replication/physiologyABSTRACT
Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and alpha2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the alpha6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, alpha2, and alpha6) are critical for the protein's ability to interact with cellular proteins and to block apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Carrier Proteins/metabolism , Caspases/metabolism , Molluscum contagiosum virus/metabolism , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Carrier Proteins/antagonists & inhibitors , Caspase 8 , Caspase 9 , Cell Line , Conserved Sequence , Fas-Associated Death Domain Protein , HeLa Cells , Humans , Jurkat Cells , Membrane Glycoproteins/pharmacology , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Structure-Activity Relationship , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/pharmacology , Viral Proteins/chemistry , Viral Proteins/genetics , fas Receptor/metabolismABSTRACT
The murine gammaherpesvirus-68 (MHV-68) M11 gene encodes a protein predicted to have limited homology to the bcl-2 family of proteins. Unlike most of the other viral bcl-2 homologues, which have both BH1 and BH2 domains conserved with respect to bcl-2, the M11 protein has a BH1 domain, but apparently lacks a BH2 domain. Transfection of HeLa cells with an epitope-tagged MHV-68 M11 construct showed that the protein is predominantly located in the cytoplasm of cells. In HeLa cells, M11 inhibited apoptosis induced by anti-Fas antibody and by TNF-alpha. Thus, despite its limited conservation with respect to other bcl-2 family members, the MHV-68 M11 protein is a potent inhibitor of apoptosis.
