Mouse Cytomegalovirus Replication Research Articles

E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells.

Viruses must counteract host cell defenses to facilitate viral replication. Viruses with a narrow host range, such as the cytomegaloviruses, are unable to counteract cellular defenses in cells of a foreign species. However, little is known about the cellular host range factors restricting cytomegalovirus replication. Here, we show that mouse cytomegalovirus (MCMV) induces the expression of the FAM111 proteases and that FAM111B, but not FAM111A that has previously been shown to restrict the replication of polyomavirus and orthopoxvirus host range mutants, acts as a cellular factor suppressing MCMV replication in human and rhesus monkey cells. The identification of FAM111B as a host range factor should provide new insight into the physiological functions of this poorly characterized protein.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication.

Human cytomegalovirus (HCMV) is a prototypic betaherpesvirus which causes severe manifestations in individuals with impaired or immature immunity. To investigate cytomegalovirus-induced pathogenesis and virus-specific immune responses, mouse cytomegalovirus (MCMV) infections in mice are employed as accepted small animal model. MCMV and HCMV share co-linear genomes and encode several homologous proteins. Due to the size and complexity of CMV genomes, the molecular functions of numerous cytomegaloviral gene products remain to be elucidated. While the essential nature of viral genes highlights their biological relevance, it renders functional studies particularly cumbersome by precluding experiments in the infection context. The HCMV-encoded protein pUL34 binds the HCMV genome and regulates viral gene expression (e.g., of US3). Several groups provided compelling evidence that UL34 is essential for HCMV replication. MCMV encodes the homologous protein pM34 (34% identical and 55% similar). Based on unsuccessful attempts to reconstitute M34-deficient virus from a bacterial artificial chromosome (BAC), M34 was previously classified as essential for MCMV replication. To characterize pM34 during viral infection, we engineered and analyzed an MCMV mutant expressing an HA-epitope-tagged pM34 which was expressed with early-late kinetics and localized in the nucleus. Additionally, we generated an M34-deficient (“ΔM34”) MCMV-BAC by replacing the entire M34 coding sequence by a kanamycin resistance cassette. The deletion of M34 was confirmed by Southern blot and PCR. Unexpectedly, we could reconstitute replicating ΔM34-MCMV upon transfection of the BAC DNA into mouse embryonic fibroblasts. The absence of M34 from the genome of the replicating ΔM34-MCMV was also confirmed. Accordingly, a ΔM34-MCMV, in which the kanamycin cassette was excised by frt/Flp-mediated recombination, was also replication competent. In order to corroborate the absence of pM34 protein, the M34 deletion was recapitulated on the background of M34HA, which yielded replicating virus devoid of detectable pM34HA protein. The replication of MCMVs lacking M34 was found to be 10- to 100-fold reduced as compared to wt-MCMV which might explain previous unsuccessful reconstitution attempts conducted by others. Taken together, our findings reveal that MCMV remains replication competent despite the absence of M34, enabling functional studies in the infection context.

Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range.

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells.

STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist.

ABSTRACTA pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo. In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCE The host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virus in vitro and in vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibility in vitro and pronounced attenuation in vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

Viral Replication Assay in Bone Marrow-Derived Macrophages.

The selection of macrophages as a cell type for investigating virus-host interactions is based on cellular tropism of the virus during infection as well as contribution of these cells to pathogenesis in the host. In response to mouse cytomegalovirus (MCMV) infection, bone marrow-resident monocytes that mobilize to infected tissues to differentiate into macrophages and dendritic cells are hijacked in order to facilitate viral persistence. These cells contribute significantly to MCMV biology and, thus, are actively recruited by the virus-encoded chemokine. In this chapter, we provide detailed methodologies employed in our laboratory to assess MCMV replication in bone marrow-derived macrophages.

Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes.

The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-α) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-γ. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells. Cytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

Cytomegalovirus-Specific CD4 T Cells Are Cytolytic and Mediate Vaccine Protection.

CD4 T cells provide protection against cytomegalovirus (CMV) and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here, we report the first two I-A(d)-restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes and use a major histocompatibility complex class II (MHC-II) tetramer to characterize their phenotypes and functions. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope- and organ-specific manner. In addition, CD4 T cell epitope vaccination of immunocompetent mice reduced MCMV replication in the same organs where CD4 cytotoxic T lymphocyte (CTL) activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo. CD4 T cells mediate immune protection by using their T cell receptors to recognize specific portions of viral proteins, called epitopes, that are presented by major histocompatibility complex class II (MHC-II) molecules on the surfaces of professional antigen-presenting cells (APCs). In this study, we discovered the first two epitopes derived from mouse cytomegalovirus (MCMV) that are recognized by CD4 T cells in BALB/c mice, a mouse strain commonly used to study the pathogenesis of this virus infection. Here, we report the sequences of these epitopes, characterize the CD4 T cells that recognize them to fight off MCMV infection, and show that we can use the epitopes to vaccinate mice and protect against MCMV.

Dissecting the Requirements for Maintenance of the CMV-Specific Memory T-Cell Pool

Cytomegalovirus (CMV) infection promotes a broad T-cell response, with the resulting memory cells displaying diverse phenotypes. CMV establishes lifelong persistence/latency, and it is thought that viral antigens expressed during this period may regulate the expansion and/or maintenance of "inflationary" CD8 T-memory populations that display an effector memory phenotype. We show here that mouse CMV (MCMV)-specific inflationary memory T cells do not decrease in number after thymectomy, indicating that recent thymic emigrants are not strictly required for their maintenance. Furthermore, persistent MCMV replication in the salivary gland does not significantly impact the T-cell memory compartment, as surgical removal did not alter its composition. These results shed light upon the mechanisms required for maintenance of the large, MCMV-specific T-cell memory pool.

Natural killer cells in NOD.NK1.1 mice acquire cytolytic function during viral infection and provide protection against cytomegalovirus

Resting natural killer (NK) cells in nonobese diabetic (NOD) mice have impaired immune functions compared with NK cells from other mouse strains. Here we investigated how NOD NK cells respond after mouse cytomegalovirus (MCMV) infection, using NOD mice congenic for the protective NK gene complex from C57BL/6 mice. Compared with C57BL/6 mice congenic for the H2 gene complex from NOD mice (B6.g7), NOD.NK1.1 mice fail to control early infection with MCMV. After MCMV infection, however, NOD.NK1.1 NK cells demonstrate increased cytolytic function, associated with higher expression of granzyme B, and undergo robust expansion. One week after infection, NOD.NK1.1 NK cells control MCMV replication as effectively as B6.g7 NK cells, even in the absence of T cells and B cells. Thus, the impaired cytotoxic function of NK cells in NOD mice is alleviated by viral infection, which enables NOD NK cells to efficiently control MCMV infection.

Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by approximately 100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.

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

We have investigated beta interferon (IFN-beta) and IFN-alpha4 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-beta and IFN-alpha4 gene expression. The induction of IFN transcription resulted from the activation of the components of the IFN-beta enhanceosome, i.e. IFN regulatory factor (IRF) 3, nuclear factor (NF)-kappaB, 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-alpha/beta 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 IkappaBalpha 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.

Effects of a selective COX-2 inhibitor (MF-tryciclic) on atherosclerosis progression as well as on mouse cytomegalovirus replication in ApoE knockout mice

Effects of a selective COX-2 inhibitor (MF-tryciclic) on atherosclerosis progression as well as on mouse cytomegalovirus replication in ApoE knockout mice

Suppression of high mobility group protein T160 expression impairs mouse cytomegalovirus replication.

The high mobility group (HMG-1) box proteins bind both non-B-DNA conformations and specific nucleotide sequences. They have been implicated in a wide variety of cellular functions involving DNA, such as transcription, replication and recombination. To determine whether HMG-1 box protein T160 plays a role in virus replication, we employed an antisense strategy to inhibit its expression in NIH 3T3 cells. The two T160 clones that expressed levels of T160 50% lower than those expressed by clones transfected with the empty vector (Neo+ clones) were investigated with respect to their permissiveness to the growth of viruses representing three families: Rhabdoviridae, vesicular stomatitis virus (VSV); Picornaviridae, encephalomyocarditis virus (EMCV), and Alpha- and Betaherpesviridae, herpes simplex virus 1 (HSV-1) and mouse cytomegalovirus (MCMV), respectively. They displayed a high degree of resistance to MCMV replication, but were fully permissive to the other viruses. Competitive PCR and probing IE-1 products by Western blot analysis showed that this resistance was not due to depressed levels of virus adsorption during the early phases of infection. We therefore conclude that T160 is involved in replication of the betaherpesvirus MCMV.