Mouse cytomegalovirus inhibits beta interferon (IFN-β) gene expression and controls activation pathways of the IFN-β enhanceosome

Abstract

We have investigated beta interferon (IFN-b) and IFN-a4 gene expression and activation of related transcription factors in mouse cytomegalovirus (MCMV)-infected fibroblasts. mRNA analysis demonstrated an initial phase of IFN gene induction upon MCMV infection, which was followed by a sustained MCMV-mediated simultaneous downregulation of IFN-b and IFN-a4 gene expression. The induction of IFN transcription resulted from the activation of the components of the IFN-b enhanceosome, i.e. IFN regulatory factor (IRF) 3, nuclear factor (NF)-kB, activating transcription factor (ATF)-2 and c-Jun. Activation of the transcription factors occurred rapidly and in a sequential order upon infection, but only lasted a while. As a consequence, IFN-a/b gene expression became undetectable 6 h post-infection and throughout the MCMV replication cycle. This effect is based on an active interference since restimulation of IFN gene induction by further external stimuli (e.g. Sendai virus infection) was completely abolished. This inhibition required MCMV gene expression and was not observed in cells infected with UV-inactivated MCMV virions. The efficiency of inhibition is achieved by a concerted blockade of IkBa degradation and a lack of nuclear accumulation of IRF3 and ATF-2/c-Jun. Using an MCMV mutant lacking pM27, a signal transducer and activator of transcription (STAT) 2-specific inhibitor of Jak/STAT signalling, we found that the initial phase of IFN induction and the subsequent inhibition does not depend on the positive-IFN feedback loop. Our findings indicate that the MCMV-mediated downregulation of IFN transcription in fibroblasts relies on a large arsenal of inhibitory mechanisms targeting each pathway that contributes to the multiprotein enhanceosome complex. INTRODUCTIONUpon virus infection the type I interferon (IFN-a/b) response is one of the earliest innate host defence mechanisms, and many viruses have found means to avoid IFN gene expression Haller et al., 2006). Production of IFN-a/b is induced by the recognition of pathogen-associated molecular patterns (PAMP) (Medzhitov & Janeway, 2002) and initiates transcriptional upregulation of IFN-stimulated genes (ISGs) to establish an antiviral state. IFN-a/b synthesized by virus-infected cells bind to the IFN-a/b receptor complex, termed IFNAR, and activates the Jak/STAT signal transduction cascade, leading to the formation of the IFN-stimulated gene factor 3 (ISGF3). ISGF3 binds to IFN-stimulated response elements (ISRE), located in the promoter region of IFNinducible target genes mediating IFN effector functions (Darnell et al., 1994).Initiation of IFN transcription and its subsequent autocrine and paracrine amplification is a prerequisite for efficient IFN effector responses. IFN-b gene transcription is controlled by a higher order transcription enhancer complex, known as enhanceosome (Maniatis, 1986;Maniatis et al., 1998). The multi-component complex includes three distinct transcription factors binding cooperatively to the IFN-b promoter and the architectural high mobili...

Gamma Interferon-Induced Interferon Regulatory Factor 1-Dependent Antiviral Response Inhibits Vaccinia Virus Replication in Mouse but Not Human Fibroblasts

“…Expression analysis of viral transcripts by semiquantitative reverse transcriptase PCR (RT-PCR) was done as described previously (38). Briefly, total RNA was extracted using an RNeasy minikit (Qiagen).…”

“…The RNA was digested with DNase I before semiquantitative RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) with serial 10-fold dilutions (as indicated) of total RNA as a template. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase)-specific primer set has been described previously (38). The following primers were used to amplify the known vaccinia virus early gene C11R coding for the vaccinia virus growth factor (72) and the known late gene F17R (77): VACVearly (C11R)_forw (5Ј-CATTCGCCGATAGTGGTAACGC-3Ј), VACVearly (C11R)_rev (5Ј-ATCTCCCTCTGGACCGCAT-3Ј), VACVlate (F17R)_forw (5Ј-AGATAAACCCTCATCGCCCGC-3Ј), and VACVlate(F17R)_rev (5Ј-CACGTTGTCGCGATTAGCCG-3Ј).…”

Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation

“…Experimental studies using MCMV indicate that IFN receptor-induced Jak-STAT signalling has an indispensable role in the control of CMV replication by the host immune system (Lucin et al, 1992(Lucin et al, , 1994Heise et al, 1998;Presti et al, 1998). By replicating under the selective pressure of IFNs, CMVs have evolved effective mechanisms that counteract IFN induction (Le et al, 2008) and IFN-mediated antiviral defence mechanisms Zimmermann & Hengel, 2006).…”

“…Experimental studies using MCMV indicate that IFN receptor-induced Jak-STAT signalling has an indispensable role in the control of CMV replication by the host immune system (Lucin et al, 1992(Lucin et al, , 1994Heise et al, 1998;Presti et al, 1998). By replicating under the selective pressure of IFNs, CMVs have evolved effective mechanisms that counteract IFN induction (Le et al, 2008) and IFN-mediated antiviral defence mechanisms Zimmermann & Hengel, 2006).CMVs are able to interfere with IFN-dependent signal transduction in infected cells. STAT1 tyrosine phosphorylation and nuclear translocation are blocked upon HCMV infection (Miller et al, 1998; Le Roy et al, 1999) by an HCMV-induced decrease in Jak1 levels (Miller et al, 1998) which also impedes IFN-a/b-dependent signal transduction (Miller et al, 1999).…”

Murine Cytomegalovirus Virion-Associated Protein M45 Mediates Rapid NF-κB Activation after Infection

“…Also like HCMV, MCMV induces a transient phase of NF-B activation within the first few hours of infection (29,30). However, the viral proteins and cellular signaling pathways involved have not been defined.…”