Murine Cytomegalovirus Replication Research Articles

Viral infection causes long term changes of the immunological milieu within the heart

Abstract Background The last two decades have brought plentiful insights and new advances in understanding virus-host interactions, and the importance of acute and latent viral infections in the modulation of the structure and function of the mammalian immune system became apparent. Despite abundant evidence demonstrating substential changes after viral infections in the immune cell composition and the local immune response within various organs such as the lungs and the gut, the same phenomena within the heart tissue has remained poorly investigated. Methods C57BL6/J 6–12-week-old female mice were infected intravenously with murine cytomegalovirus (MCMV) or left uninfected. Kinetics of viral replication within the heart were determined using a standard plaque assay. Furthermore, immune cell composition within the heart was determined by flow cytometry during acute and latent viral infection. Results Active MCMV replication within the heart peaked at day 5 post-infection (p.i.) and the replicating viral particles became undetectable by day 12 in all of the infected animals. The initial immune cell infiltration was led by the increase in the NK cell compartment, followed by a substantial increase in the T cell compartment which peaked around day 7 p.i. Furthermore, there were substantial phenotypic changes in the population of cardiac macrophages which became predominantly MHCIIhigh and Ly6Chigh. Most notable was an enlargement within the CD8 T cell population which increased around 60-fold compared to the steady state conditions. Crucially, the changes in the immune cell milieu were maintained long after cessation of active viral replication as the hearts of latently infected mice contained around 2.5 times more leukocytes (CD45+) than the hearts of non-infected animals, around 18% percent of which were CD8 T cells, compared to 5% observed in the mock-infected group. Conclusion Systemic viral infection with a ubiquitous herpesvirus causes long term alterations in the composition and phenotype of the heart immune cells which could have major consequences for myocardial coping with different pathological injuries such as a subsequent microbial infection, ischemic conditions or stress induced by hypertension or volume overload.

Fibroblasts are a site of murine cytomegalovirus lytic replication and Stat1-dependent latent persistence in vivo

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a β-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

A defined anthocyanin mixture sourced from bilberry and black currant inhibits Measles virus and various herpesviruses

BackgroundAnthocyanin-containing plant extracts and carotenoids, such as astaxanthin, have been well-known for their antiviral and anti-inflammatory activity, respectively. We hypothesised that a mixture of Ribes nigrum L. (Grossulariaceae) (common name black currant (BC)) and Vaccinium myrtillus L. (Ericaceae) (common name bilberry (BL)) extracts (BC/BL) with standardised anthocyanin content as well as single plant extracts interfered with the replication of Measles virus and Herpesviruses in vitro.MethodsWe treated cell cultures with BC/BL or defined single plant extracts, purified anthocyanins and astaxanthin in different concentrations and subsequently infected the cultures with the Measles virus (wild-type or vaccine strain Edmonston), Herpesvirus 1 or 8, or murine Cytomegalovirus. Then, we analysed the number of infected cells and viral infectivity and compared the data to non-treated controls.ResultsThe BC/BL extract inhibited wild-type Measles virus replication, syncytia formation and cell-to-cell spread. This suppression was dependent on the wild-type virus-receptor-interaction since the Measles vaccine strain was unaffected by BC/BL treatment. Furthermore, the evidence was provided that the delphinidin-3-rutinoside chloride, a component of BC/BL, and purified astaxanthin, were effective anti-Measles virus compounds. Human Herpesvirus 1 and murine Cytomegalovirus replication was inhibited by BC/BL, single bilberry or black currant extracts, and the BC/BL component delphinidin-3-glucoside chloride. Additionally, we observed that BC/BL seemed to act synergistically with aciclovir. Moreover, BC/BL, the single bilberry and black currant extracts, and the BC/BL components delphinidin-3-glucoside chloride, cyanidin-3-glucoside, delphinidin-3-rutinoside chloride, and petunidin-3-galactoside inhibited human Herpesvirus 8 replication.ConclusionsOur data indicate that Measles viruses and Herpesviruses are differentially susceptible to a specific BC/BL mixture, single plant extracts, purified anthocyanins and astaxanthin. These compounds might be used in the prevention of viral diseases and in addition to direct-acting antivirals, such as aciclovir.

Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication

Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis.The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy.In cultured MEFs, the ratio of LC3I/II was reduced at 24 hourspost infection (hpi), but was increased at 48 hpiIn the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpiin MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups.Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.

The Role of Autophagy in Murine Cytomegalovirus Hepatitis.

Autophagy is involved in the pathogenesis of multiple pathogen infection. Previous studies have reported that human cytomegalovirus (CMV) activates autophagy in the early stage of infection and then inhibits autophagy. Little is known about the role of autophagy in murine CMV (MCMV) infection, especially in MCMV-induced hepatitis. The purpose of this study is to investigate the role of autophagy in MCMV hepatitis. BALB/c mice were infected with MCMV and a series of experiments involving western blot, immunofluorescence, immunohistochemistry, H&E (Hematoxylin and Eosin) staining and quantitative real-time polymerase chain reaction were performed in this study. The expression of SQSTM1/p62, PI3K, the ratio of phosphorylated Akt to total Akt, and the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR were increased, and the expression of light-chain 3 (LC3)-II were decreased in the livers of infected mice on days 3 and 7 postinfection (p.i.). Compared with the untreated infected group, increased transcription level of MCMV glycoprotein B (gB), increased expression levels of interleukin1-β (IL-1β), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreased expression level of type I interferon α (IFN-α), as well as aggravated liver pathological injury were detected in starvation-treated infected group on days 3 and 7 p.i.; whereas decreased transcription level of MCMV gB, decreased expression levels of IL-1β, AST and ALT, increased expression level of type I IFN-α, as well as alleviated liver pathological injury were detected in chloroquine (CQ)-treated infected group on day 3 p.i. In conclusion, autophagy is inhibited through activating the PI3K/Akt/mTOR pathway in the liver of BALB/c mice during MCMV infection, and autophagy may promote MCMV replication and aggravate liver pathological damage and inflammation. Further understanding of the interactions between autophagy and MCMV infection and its potential mechanism may bring new important cues to the control of MCMV infection and antiviral therapy.

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus.

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.

Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue.

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

Heart Dysfunction Following Long-Term Murine Cytomegalovirus Infection: Fibrosis, Hypertrophy, and Tachycardia.

Human cytomegalovirus (HCMV) is associated with increased risk of chronic diseases of the heart and vasculature, including myocarditis, atherosclerosis, and transplant vasculopathy. To investigate CMV infection of the heart, murine cytomegalovirus (MCMV) was used to evaluate both acute and latent infection and the subsequent phenotypic and functional consequences of infection. Female BALB/c mice were intraperitoneally (i.p.) inoculated with 1 × 106 pfu of MCMV and evaluated at 14 and 50 days postinfection (dpi). At each time point, echocardiography was used to evaluate cardiac function and histology was conducted for phenotypic evaluation. MCMV replication in the heart was detected as early as 3 dpi and was no longer detectable at 14 dpi. Infected animals had significant cardiac pathology at 14 and 50 dpi when compared to uninfected controls. Histology revealed fibrosis of the heart as early as 14 dpi and the presence of white fibrous deposits on the surface of the heart. Functional evaluation showed significantly increased heart rate and muscle thickening in the latently infected animals when compared to the control animals. At 50 dpi, latent virus was measured by explant reactivation assay, demonstrating that MCMV establishes latency and is capable of reactivation from the heart, similar to other tissues such as spleen and salivary glands. Collectively, these studies illustrate that MCMV infection results in phenotypic alterations within the heart as early as 14 dpi, which progress to functional abnormalities during latency. These findings are similar to sinus tachycardia and hypertrophy of the heart muscle observed in cases of HCMV-induced acute myocarditis.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

STING induces early IFN-\u03b2 in the liver and constrains myeloid cell-mediated dissemination of murine cytomegalovirus

Cytomegalovirus is a DNA-encoded β-herpesvirus that induces STING-dependent type 1 interferon responses in macrophages and uses myeloid cells as a vehicle for dissemination. Here we report that STING knockout mice are as resistant to murine cytomegalovirus (MCMV) infection as wild-type controls, whereas mice with a combined Toll-like receptor/RIG-I-like receptor/STING signaling deficiency do not mount type 1 interferon responses and succumb to the infection. Although STING alone is dispensable for survival, early IFN-β induction in Kupffer cells is STING-dependent and controls early hepatic virus propagation. Infection experiments with an inducible reporter MCMV show that STING constrains MCMV replication in myeloid cells and limits viral dissemination via these cells. By contrast, restriction of viral dissemination from hepatocytes to other organs is independent of STING. Thus, during MCMV infection STING is involved in early IFN-β induction in Kupffer cells and the restriction of viral dissemination via myeloid cells, whereas it is dispensable for survival.

Potential Application of TALENs against Murine Cytomegalovirus Latent Infections.

Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1–2, 3–4, and 5–6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.

In vivo proof-of-concept for two experimental antiviral drugs, both directed to cellular targets, using a murine cytomegalovirus model

Infections with the human cytomegalovirus (HCMV) cause serious medical problems including organ rejection and congenital infection. Treatment of HCMV infections with currently available medication targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. This demands novel therapeutical approaches like targeting genetically stable host cell proteins that are crucial for virus replication. Although numerous experimental drugs with promising in vitro efficacy have been identified, the lack of available data in animal models limits their potential for further clinical development. Recently, we described the very strong in vitro antiherpesviral activity of the NF-κB inhibitor TF27 and the CDK7 inhibitor LDC4297 at low nanomolar concentrations. In the present study, we present first data for the in vivo efficacy of both experimental drugs using an established cytomegalovirus animal model (murine CMV replication in immunodefective Rag −/− mice). The main findings of this study are (i) a strong inhibitory potency against beta- and gamma-herpesviruses of both compounds in vitro, (ii) even more important, a pronounced anticytomegaloviral activity also exerted in vivo, that resulted from (iii) a restriction of viral replication to the site of infection, thus preventing organ dissemination, (iv) in the absence of major compound-associated adverse events. Thus, we provide evidence for a strong antiviral potency in vivo and proof-of-concept for both drugs, which may encourage their further drug development, possibly including pharmacologically optimized derivatives, for a potential use in future antiherpesviral treatment.

Murine cytomegalovirus M72 promotes acute virus replication in vivo and is a substrate of the TRiC/CCT complex

Betaherpesvirus dUTPase homologs are core herpesvirus proteins, but little is known about their role during infection. Human cytomegalovirus (HCMV) UL72 and murine cytomegalovirus (MCMV) M72 have been designated dUTPase homologs, and previous studies indicate UL72 is dispensable for replication and enzymatically inactive. Here, we report the initial characterization of MCMV M72. M72 does not possess dUTPase activity, and is expressed as a leaky-late gene product with multiple protein isoforms. Importantly, M72 augments MCMV replication in vitro and during the early stage of acute infection in vivo. We identify and confirm interaction of M72 with the eukaryotic chaperonin tailless complex protein -1 (TCP-1) ring complex (TRiC) or chaperonin containing tailless complex polypeptide 1 (CCT). Accumulating biochemical evidence indicates M72 forms homo-oligomers and is a substrate of TRiC/CCT. Taken together, we provide the first evidence of M72's contribution to viral pathogenesis, and identify a novel interaction with the TRiC/CCT complex.

Correction for Perez et al., "A Short cis-Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication".

Correction for Perez et al., "A Short cis-Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication".

Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity.

The HIRA histone chaperone complex deposits histone H3.3 into nucleosomes in a DNA replication- and sequence-independent manner. As herpesvirus genomes enter the nucleus as naked DNA, we asked whether the HIRA chaperone complex affects herpesvirus infection. After infection of primary cells with HSV or CMV, or transient transfection with naked plasmid DNA, HIRA re-localizes to PML bodies, sites of cellular anti-viral activity. HIRA co-localizes with viral genomes, binds to incoming viral and plasmid DNAs and deposits histone H3.3 onto these. Anti-viral interferons (IFN) specifically induce HIRA/PML co-localization at PML nuclear bodies and HIRA recruitment to IFN target genes, although HIRA is not required for IFN-inducible expression of these genes. HIRA is, however, required for suppression of viral gene expression, virus replication and lytic infection and restricts murine CMV replication in vivo. We propose that the HIRA chaperone complex represses incoming naked viral DNAs through chromatinization as part of intrinsic cellular immunity.

Lacrimal Gland NK Cells Are Developmentally and Functionally Similar to Conventional NK Cells.

The murine lacrimal gland (LG), which produces crucial components of the ocular tear film, contains a population of natural killer (NK) cells. LG NK cells appear to belong to the conventional NK cell lineage, based on their cell surface receptor and transcription factor expression, absence in NFIL3-/- mice, and lack of RORγt expression during development. LG NK cells produce IFN-γ during the early stages of systemic murine cytomegalovirus (MCMV) infection. This effector response occurs in the absence of noticeable MCMV replication in the LG, indicating that LG NK cells are being activated by soluble factors. However, the magnitude of LG NK cell IFN-γ production during MCMV infection is significantly lower than spleen and liver NK cells. Adoptive transfer experiments in lymphopenic mice revealed that this hyporesponsive phenotype is tissue-specific, which indicates that that LG NK cells can produce a robust effector response.

The antiviral restriction factor IFN-induced transmembrane protein 3 prevents cytokine-driven CMV pathogenesis.

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis.

Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. Herpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced, leading to the expression of several proteins from a single gene. In this study, we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have nonredundant functions.

Functional Comparison of Molluscum Contagiosum Virus vFLIP MC159 with Murine Cytomegalovirus M36/vICA and M45/vIRA Proteins.

Molluscum contagiosum virus (MCV) gene MC159 encodes a viral FLICE inhibitory protein (vFLIP) that inhibits caspase-8-mediated apoptosis. The MC159 protein was also reported to inhibit programmed necrosis (necroptosis) and modulate NF-κB activation by interacting with RIP1 and NEMO. The importance of MC159 during MCV infection has remained unknown, as there is no system for propagation and genetic manipulation of this virus. Here we investigated the functions of MC159 during viral infection using murine cytomegalovirus (MCMV) as a surrogate virus. MC159 was inserted into the MCMV genome, replacing M36 or M45, two MCMV genes with functions similar to those reported for MC159. M36 encodes a viral inhibitor of caspase-8-induced apoptosis (vICA) and M45 a viral inhibitor of RIP activation (vIRA), which inhibits RIP1/RIP3-mediated necroptosis. The M45 protein also blocks NF-κB activation by interacting with NEMO. When expressed by MCMV, MC159 blocked tumor necrosis factor alpha (TNF-α)-induced apoptosis of infected cells and partially restored MCMV replication in macrophages. However, MC159 did not fully replace M45, as it did not inhibit necroptosis in murine cells, but it reduced TNF-α-induced necroptosis in MCMV-infected human HT-29 cells. MC159 also differed from M45 in its effect on NF-κB. While MCMV-encoded M45 blocked NF-κB activation by TNF-α and interleukin-1β (IL-1β), MC159 inhibited TNF-α- but not IL-1β-induced NF-κB activation in infected mouse fibroblasts. These results indicate that the spectrum of MC159's functions differs depending on cell type and expression system and that a cell culture system for the propagation of MCV is needed to determine the biological relevance of presumed viral gene functions. MCV is a human-pathogenic poxvirus that cannot be propagated in cell culture or laboratory animals. Therefore, MCV gene products have been studied predominantly in cells expressing individual viral genes. In this study, we analyzed the function of the MCV gene MC159 by expressing it from a different virus and comparing its functions to those of two well-characterized MCMV genes. In this system, MC159 displayed some but not all of the previously described functions, suggesting that the functions of a viral gene depend on the conditions under which it is expressed. Until a cell culture system for the analysis of MCV becomes available, it might be necessary to analyze MCV genes in several different systems to extrapolate their biological importance.

ID: 170: IFITM3 restricts virus-induced pathogenesis by regulating myeloid cell production of interleukin-6

The antiviral restriction factor interferon-induced transmembrane protein 3 (IFITM3) inhibits endocytosis-dependent entry of numerous viruses and determines susceptible to viral disease in humans. Using the murine cytomegalovirus (MCMV) model, we discovered that regulation of pro-inflammatory cytokine production is a critical function of IFITM3. IFITM3 prevented MCMV-induced weight loss and death without directly restricting MCMV replication. Instead, susceptibility of IFITM3−/− mice was attributed to an enhanced MCMV-induced lymphopenia, activation-induced NK cell death and impaired T cell responses, resulting in uncontrolled virus replication. Exacerbated pathology in IFITM3−/− mice was associated with increased expression of the pro-inflammatory cytokine interleukin-6 (IL-6), and IFITM3 exerted cell-intrinsic inhibition of MCMV-induced IL-6 expression by myeloid cells. Although IL-6 is required for the generation of antiviral adaptive immunity, inhibiting anti-IL-6R reversed MCMV-induced pathology and control of early MCMV replication. These data suggest that IFITM3 restricts viral pathogenesis, at least in part, via regulation of pro-inflammatory cytokine production.