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1.
J Virol ; 96(12): e0041922, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35638820

RESUMO

Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro. IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro, we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo. We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro. mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Proteínas de Resistência a Myxovirus , Replicação Viral , Animais , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Interferons/metabolismo , Camundongos , Muromegalovirus , Proteínas de Resistência a Myxovirus/metabolismo
2.
BMC Immunol ; 23(1): 17, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35439922

RESUMO

BACKGROUND: Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection. RESULTS: Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells. CONCLUSIONS: These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.


Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Linfócitos T CD8-Positivos , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/metabolismo , Imunidade , Células Matadoras Naturais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologia
3.
J Immunol ; 208(7): 1742-1754, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35321880

RESUMO

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.


Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo
4.
Exp Eye Res ; 218: 109009, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35276185

RESUMO

Accumulated clinical evidence has shown that Posner-Schlossman syndrome (PSS) is most likely the result of recurrent human cytomegalovirus (HCMV) infection in the anterior chamber (AC). Establishing an animal model is necessary to investigate the pathogenesis of PSS. In this study, we constructed a mouse model of (PSS) by injecting murine cytomegalovirus (MCMV) into the AC of BALB/c mice. Twenty-five BALB/c mice were divided into 5 groups. Smith strain MCMV expressing enhanced green fluorescent protein (EGFP) was passaged with mouse embryonic fibroblast (MEF). Right eyes in the 4 experiment groups received AC injection of 1 µL of virus solution with concentrations of 103,104,105,106 pfu/mL respectively, and the control group received only PBS. PSS-like signs (mutton-fat keratic precipitates (KP), pupil dilation, IOP elevation and corneal edema) were recorded 0-28 days post-injection (DPI). Sections of eyeballs from another 9 mice harvested on 0,10 and 28 DPI were examined to locate KP and the fluorescence signal of the virus. Reversible PSS-like signs except KP were observed in 20% and 60% mice of 104 and 105 groups while no PSS-like signs in the control and 103 group; 80% in the 106 group with partially unreversible signs till 28DPI. Much More fluorescent signals of virus in the iris and KP were found on 10DPI than 28 DPI, while no fluorescent signals and KP on 0DPI. The extent of PSS-like signs (pupil dilation, IOP elevation and corneal edema) was virus concentration-dependent (Spearman correlation coefficient, r = 0.830, = 0.475, = 0.662, p < 0.0001, <0.05, <0.001, respectively, n = 25). Success rate of PSS model (mice with PSS-like signs) was also virus concentration-dependent (Chi-square trend test, χ2 = 6.828, df = 1, p < 0.01, n = 25). Our results indicate that AC injection of 1 µL MEF passaged MCMV (Smith strain) of 104-106 pfu/mL in BALB/c mice can be used to construct a mouse model of PSS. MCMV can infect iris tissue and replicate in it and then establish latency. This might account for the recurrent and self-limited nature of PSS.


Assuntos
Edema da Córnea , Infecções por Citomegalovirus , Glaucoma de Ângulo Aberto , Muromegalovirus , Animais , Câmara Anterior/patologia , Citomegalovirus , Modelos Animais de Doenças , Fibroblastos/patologia , Camundongos , Camundongos Endogâmicos BALB C
5.
Methods Mol Biol ; 2463: 195-204, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35344176

RESUMO

Immunological memory is a fundamental feature of the adaptive immune system that protects the host from recurrent infections from pathogens. Natural killer (NK) cells are a predominant member of the innate immune system that lack clonotypic receptors, which are essential for memory formation. However, evidence demonstrates that a unique subpopulation of NK cells develops adaptive-like features using germline-encoded receptors. Recent studies have shown that infection of cytomegalovirus (CMV) leads to clonal expansion of NKG2C+ and Ly49H+ NK cells, in humans and mouse, respectively. These activation receptors have the capability to recognize CMV-encoded proteins and facilitate a recall response upon reinfection. Although NK cells do not rearrange genes encoding their activating receptors as seen in B and T cells, they possess a selective process to generate memory features and a long-lived progeny. Here, we describe an established in vivo protocol for infecting mice with mouse cytomegalovirus (MCMV) to study an adaptive NK cell response.


Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Citomegalovirus , Memória Imunológica , Células Matadoras Naturais , Camundongos
6.
J Virol ; 96(7): e0007722, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35293772

RESUMO

CD4+ T cells are key to controlling cytomegalovirus infections. Salivary gland infection by murine cytomegalovirus (MCMV) provides a way to identify mechanisms. CD11c+ dendritic cells (DC) disseminate MCMV to the salivary glands, where they transfer infection to acinar cells. Antiviral CD4+ T cells are often considered to be directly cytotoxic for cells expressing major histocompatibility complex class II (MHCII). However, persistently infected salivary gland acinar cells are MHCII- and are presumably inaccessible to direct CD4 T cell recognition. Here, we show that CD4+ T cell depletion amplified infection of MHCII- acinar cells but not MHCII+ cells. MCMV-infected mice with disrupted MHCII on CD11c+ cells showed increased MHCII- acinar infection; antiviral CD4+ T cells were still primed, but their recruitment to the salivary glands was reduced, suggesting that engagement with local MHCII+ DC is important for antiviral protection. As MCMV downregulates MHCII on infected DC, the DC participating in CD4 protection may thus be uninfected. NK cells and gamma interferon (IFN-γ) may also contribute to CD4+ T cell-dependent virus control: CD4 T cell depletion reduced NK cell recruitment to the salivary glands, and both NK cell and IFN-γ depletion equalized infection between MHCII-disrupted and control mice. Taken together, these results suggest that CD4+ T cells protect indirectly against infected acinar cells in the salivary gland via DC engagement, requiring the recruitment of NK cells and the action of IFN-γ. Congruence of these results with an established CD4+ T cell/NK cell axis of gammaherpesvirus infection control suggests a common mode of defense against evasive viruses. IMPORTANCE Cytomegalovirus infections commonly cause problems in immunocompromised patients and in pregnancy. We lack effective vaccines. CD4+ T cells play an important role in normal infection control, yet how they act has been unknown. Using murine cytomegalovirus as an accessible model, we show that CD4+ T cells are unlikely to recognize infected cells directly. We propose that CD4+ T cells interact with uninfected cells that present viral antigens and recruit other immune cells to attack infected targets. These data present a new outlook on understanding how CD4+ T cell-directed control protects against persistent cytomegalovirus infection.


Assuntos
Linfócitos T CD4-Positivos , Infecções por Citomegalovirus , Muromegalovirus , Animais , Antivirais , Linfócitos T CD4-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Humanos , Interferon gama , Camundongos , Muromegalovirus/imunologia
7.
Nat Immunol ; 23(4): 556-567, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35288713

RESUMO

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single-cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus infection. Ly6C- memory precursor (MP) NK cells showed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. MP NK cells exhibited distinct transcriptional and epigenetic signatures compared with Ly6C+ NKeff cells, with a core epigenetic signature shared with MP CD8+ T cells enriched in ETS1 and Fli1 DNA-binding motifs. Fli1 was induced by STAT5 signaling ex vivo, and increased levels of the pro-apoptotic factor Bim in early effector NK cells following viral infection. These results suggest that a NK cell-intrinsic checkpoint controlled by the transcription factor Fli1 limits MP NK formation by regulating early effector NK cell fitness during viral infection.


Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Imunidade Adaptativa , Animais , Linfócitos T CD8-Positivos , Memória Imunológica , Células Matadoras Naturais , Camundongos
8.
Viruses ; 14(2)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35215828

RESUMO

Treatment options for human cytomegalovirus (CMV) remain limited and are associated with significant adverse effects and the selection of resistant CMV strains in transplant recipients and congenitally infected infants. Although most approved drugs target and inhibit the CMV DNA polymerase, additional agents with distinct mechanisms of action are needed for the treatment and prevention of CMV. In a large high throughput screen using our CMV-luciferase reporter Towne, we identified several unique inhibitors of CMV replication. Here, we synthesize and test in vitro 13 analogs of the original NCGC2955 hit (1). Analogs with no activity against the CMV-luciferase at 10 µM and 30 µM (2-6, 10-14) were removed from further analysis. Three analogs (7-9) inhibited CMV replication in infected human foreskin fibroblasts. The EC50 of (1) was 1.7 ± 0.6 µM and 1.99 ± 0.15 µM, based on luciferase and plaque assay, respectively. Compounds 7, 8, and 9 showed similar activities: the EC50 values of 7 were 0.21 ± 0.06 µM (luciferase) and 0.55 ± 0.06 (plaque), of 8: 0.28 ± 0.06 µM and 0.42 ± 0.07, and of 9: 0.30 ± 0.05 µM (luciferase) and 0.35 ± 0.07 (plaque). The CC50 for 7, 8, and 9 in non-infected human foreskin fibroblasts was > 500µM, yielding a selectivity index of >1500. Compounds 1, 7, and 8 were also tested in CMV-infected primary human hepatocytes and showed a dose-response against CMV by luciferase activity and viral protein expression. None of the active compounds inhibited herpes simplex virus 1 or 2. Compounds 7 and 8 inhibited mouse CMV replication in vitro. Both inhibited CMV at late stages of replication; 7 reduced virus yield at all late time points, although not to the same degree as letermovir. Finally, the activity of analog 8 was additive with newly identified CMV inhibitors (MLS8969, NFU1827, MSL8554, and MSL8091) and with ganciclovir. Further structural activity development should provide promising anti-CMV agents for use in clinical studies.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Animais , Células Cultivadas , Citomegalovirus/fisiologia , Ganciclovir/farmacologia , Hepatócitos/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Muromegalovirus/efeitos dos fármacos , Relação Estrutura-Atividade , Carga Viral , Replicação Viral/efeitos dos fármacos
9.
J Immunol ; 208(4): 799-806, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35091435

RESUMO

The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8+ T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8+ T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8+ T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8+ T cells are very similar to those of bulk memory-phenotype CD8+ T cells. Even CMV-specific inflationary CD8+ T cell responses that differ 3-fold in size were found to turn over at similar rates.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , /metabolismo , Muromegalovirus/imunologia , Algoritmos , Animais , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , Infecções por Citomegalovirus/virologia , Epitopos de Linfócito T/imunologia , Feminino , Imunofenotipagem , Camundongos , Modelos Teóricos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
10.
Immunohorizons ; 6(1): 8-15, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35031582

RESUMO

NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of "Ag-specific" NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains that exhibit broad genetic diversity across strains. Our assessment identified and validated a putative "ideal" CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57BL/6 mice, are capable of developing enhanced NK cell-mediated protective responses to murine CMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models.


Assuntos
Modelos Animais de Doenças , Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Transferência Adotiva , Animais , Feminino , Infecções por Herpesviridae/patologia , Células Matadoras Naturais/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
11.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35046024

RESUMO

Transmissible vaccines have the potential to revolutionize how zoonotic pathogens are controlled within wildlife reservoirs. A key challenge that must be overcome is identifying viral vectors that can rapidly spread immunity through a reservoir population. Because they are broadly distributed taxonomically, species specific, and stable to genetic manipulation, betaherpesviruses are leading candidates for use as transmissible vaccine vectors. Here we evaluate the likely effectiveness of betaherpesvirus-vectored transmissible vaccines by developing and parameterizing a mathematical model using data from captive and free-living mouse populations infected with murine cytomegalovirus (MCMV). Simulations of our parameterized model demonstrate rapid and effective control for a range of pathogens, with pathogen elimination frequently occurring within a year of vaccine introduction. Our results also suggest, however, that the effectiveness of transmissible vaccines may vary across reservoir populations and with respect to the specific vector strain used to construct the vaccine.


Assuntos
Betaherpesvirinae/genética , Vetores Genéticos/genética , Imunogenicidade da Vacina , Modelos Teóricos , Vacinas/imunologia , Algoritmos , Doenças dos Animais/prevenção & controle , Doenças dos Animais/transmissão , Doenças dos Animais/virologia , Animais , Teorema de Bayes , Reservatórios de Doenças , Vetores de Doenças , Vetores Genéticos/imunologia , Infecções por Herpesviridae/veterinária , Camundongos , Muromegalovirus , Prevalência , Vacinas/genética
12.
Viruses ; 14(1)2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35062332

RESUMO

Cytomegaloviruses (CMVs) are host species-specific and have adapted to their respective mammalian hosts during co-evolution. Host-adaptation is reflected by "private genes" that have specialized in mediating virus-host interplay and have no sequence homologs in other CMV species, although biological convergence has led to analogous protein functions. They are mostly organized in gene families evolved by gene duplications and subsequent mutations. The host immune response to infection, both the innate and the adaptive immune response, is a driver of viral evolution, resulting in the acquisition of viral immune evasion proteins encoded by private gene families. As the analysis of the medically relevant human cytomegalovirus by clinical investigation in the infected human host cannot make use of designed virus and host mutagenesis, the mouse model based on murine cytomegalovirus (mCMV) has become a versatile animal model to study basic principles of in vivo virus-host interplay. Focusing on the immune evasion of the adaptive immune response by CD8+ T cells, we review here what is known about proteins of two private gene families of mCMV, the m02 and the m145 families, specifically the role of m04, m06, and m152 in viral antigen presentation during acute and latent infection.


Assuntos
Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Muromegalovirus/genética , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Evasão da Resposta Imune , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Virais
13.
J Virol ; 96(4): e0186721, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34878888

RESUMO

Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.


Assuntos
Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/virologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Infecções por Herpesviridae/metabolismo , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Muromegalovirus/metabolismo , Mutação , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas G/genética , Glândulas Salivares/virologia , Transdução de Sinais , Proteínas Virais/genética , Viremia/metabolismo , Viremia/virologia , Ativação Viral/genética
14.
J Virol ; 96(2): e0087621, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34705561

RESUMO

Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.


Assuntos
Sistema Fagocitário Mononuclear/virologia , Muromegalovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/virologia , Glicosilação , Infecções por Herpesviridae/virologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Sistema Fagocitário Mononuclear/metabolismo , Transcrição Genética , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Montagem de Vírus , Internalização do Vírus , Replicação Viral
15.
J Virol Methods ; 301: 114436, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34929204

RESUMO

BACKGROUND: Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software. Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency. METHODS: mCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM. RESULTS: We found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue. CONCLUSIONS: For viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.


Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Citomegalovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Transcriptoma
16.
Commun Biol ; 4(1): 1355, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857864

RESUMO

Our understanding of the composition and functions of splenic stromal cells remains incomplete. Here, based on analysis of over 20,000 single cell transcriptomes of splenic fibroblasts, we characterized the phenotypic and functional heterogeneity of these cells in healthy state and during virus infection. We describe eleven transcriptionally distinct fibroblastic cell clusters, reassuring known subsets and revealing yet unascertained heterogeneity amongst fibroblasts occupying diverse splenic niches. We further identify striking differences in innate immune signatures of distinct stromal compartments in vivo. Compared to other fibroblasts and to endothelial cells, Ly6C+ fibroblasts of the red pulp were selectively endowed with enhanced interferon-stimulated gene expression in homeostasis, upon systemic interferon stimulation and during virus infection in vivo. Collectively, we provide an updated map of fibroblastic cell diversity in the spleen that suggests a specialized innate immune function for splenic red pulp fibroblasts.


Assuntos
Fibroblastos/metabolismo , Infecções por Herpesviridae/virologia , Imunidade Inata , Transcriptoma , Animais , Feminino , Fibroblastos/imunologia , Homeostase , Masculino , Camundongos , Muromegalovirus/fisiologia , Análise de Célula Única , Baço/imunologia , Baço/metabolismo
17.
Viruses ; 13(12)2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34960750

RESUMO

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.


Assuntos
Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/isolamento & purificação , Muromegalovirus/crescimento & desenvolvimento , Muromegalovirus/isolamento & purificação , Animais , Linhagem Celular , Cromatografia por Troca Iônica , Criopreservação , Exossomos , Humanos , Camundongos , Ultracentrifugação , Cultura de Vírus
18.
mBio ; 12(6): e0293421, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903047

RESUMO

Cytomegaloviruses (CMVs) are among the largest pathogenic viruses in mammals. To enable replication of their long double-stranded DNA genomes, CMVs induce profound changes in cell cycle regulation. A hallmark of CMV cell cycle control is the establishment of an unusual cell cycle arrest at the G1/S transition, which is characterized by the coexistence of cell cycle stimulatory and inhibitory activities. While CMVs interfere with cellular DNA synthesis and cell division, they activate S-phase-specific gene expression and nucleotide metabolism. This is facilitated by a set of CMV gene products that target master regulators of G1/S progression such as cyclin E and A kinases, Rb-E2F transcription factors, p53-p21 checkpoint proteins, the APC/C ubiquitin ligase, and the nucleotide hydrolase SAMHD1. While the major themes of cell cycle regulation are well conserved between human and murine CMVs (HCMV and MCMV), there are considerable differences at the level of viral cell cycle effectors and their mechanisms of action. Furthermore, both viruses have evolved unique mechanisms to sense the host cell cycle state and modulate the infection program accordingly. This review provides an overview of conserved and divergent features of G1/S control by MCMV and HCMV.


Assuntos
Pontos de Checagem do Ciclo Celular , Infecções por Citomegalovirus/fisiopatologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Muromegalovirus/fisiologia , Animais , Citomegalovirus/genética , Fase G1 , Humanos , Camundongos , Muromegalovirus/genética , Fase S
19.
Viruses ; 13(11)2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34834942

RESUMO

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.


Assuntos
Citomegalovirus/fisiologia , Necroptose , Proteínas Virais/metabolismo , Animais , Apoptose , Linhagem Celular , Citomegalovirus/genética , Herpesviridae/metabolismo , Herpesvirus Humano 1/metabolismo , Interações Hospedeiro-Patógeno , Camundongos , Vírus do Molusco Contagioso , Muromegalovirus/fisiologia , Necrose , Especificidade da Espécie , Proteínas Virais/genética
20.
Viruses ; 13(11)2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34835083

RESUMO

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 µM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.


Assuntos
Proteínas do Capsídeo/ultraestrutura , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/fisiologia , Animais , Capsídeo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Humanos , Camundongos , Muromegalovirus/metabolismo , Muromegalovirus/patogenicidade , Fosfoproteínas/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura , Vírion , Montagem de Vírus
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