Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 566
Filtrar
1.
mBio ; 13(3): e0083622, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35642944

RESUMO

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are associated with numerous malignancies, including B cell lymphomas and nasopharyngeal carcinoma. These viruses employ numerous molecular strategies to colonize the host, including the expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2, respectively) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. In work here, we used chimeric MHV68 viruses in an in vivo complementation system to test whether EBV EBER2 contributes to acute and/or chronic phases of infection. Expression of EBER2 derived from EBV strain B95-8 resulted in a significant expansion of latently infected B cells in vivo, which was accompanied by a decrease in virus-infected plasma cells. EBV strains typically carry one of two variants of EBER2, which differ primarily by a 5-nucleotide core polymorphism identified initially in the EBV strain M81. Strikingly, mutation of the 5 nucleotides that define this core polymorphism resulted in the loss of the infected B cell expansion and restored plasma cell infection. This work reveals that the B95-8 variant of EBER2 promotes the expansion of the latently infected B cell pool in vivo and may do so in part through inhibition of terminal differentiation. These findings provide new insight into mechanisms by which viral ncRNAs promote in vivo colonization and further and provide further evidence of the inherent tumorigenic risks associated with gammaherpesvirus manipulation of B cell differentiation. IMPORTANCE The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68, employ numerous strategies to colonize the host, including expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs ever identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. Work here reveals that an EBV EBER2 variant highly associated with B cell lymphoma promoted a significantly increased expansion of the infected B cell pool in vivo, which coincided with altered B cell differentiation. Mutation of the 5 nucleotides that define this EBER2 variant resulted in the loss of B cell expansion and normal B cell differentiation. These findings provide new insight into the mechanisms by which EBV manipulates B cells in vivo to retain infected cells in the high-risk B cell differentiation pathway where they are poised for tumorigenesis.


Assuntos
Infecções por Vírus Epstein-Barr , Gammaherpesvirinae , Herpesvirus Humano 8 , Rhadinovirus , Animais , Infecções por Vírus Epstein-Barr/genética , Gammaherpesvirinae/genética , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/genética , Humanos , Camundongos , Nucleotídeos , Polimorfismo Genético , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA Viral , Rhadinovirus/genética , Latência Viral/genética
2.
J Virol ; 96(14): e0063922, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35758659

RESUMO

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), are species-specific, ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a unique and robust polyclonal germinal center response to establish a long-term latent reservoir in memory B cells. The robust polyclonal germinal center response driven by gammaherpesvirus infection increases the risk for B cell transformation. Unsurprisingly, many gammaherpesvirus cancers are derived from germinal center or post-germinal center B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined. We previously showed that host interleukin 17 receptor A (IL-17RA) signaling promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we found that T cell-intrinsic IL-17RA signaling recapitulates some proviral aspects of global IL-17RA signaling during MHV68 infection. Specifically, we found that T cell-intrinsic IL-17RA signaling supports the MHV68-driven germinal center response, the establishment of latency in the spleen, and viral reactivation in the spleen and peritoneal cavity. Our study unveils an unexpected finding where the T cell-specific IL-17RA signaling supports the establishment of a latent reservoir of a B cell-tropic gammaherpesvirus. IMPORTANCE Gammaherpesviruses, such as human EBV, establish lifelong infection in >95% of adults and are associated with B cell lymphomas. Gammaherpesviruses usurp the germinal center response to establish latent infection, and the germinal center B cells are thought to be the target of viral transformation. We previously found that global expression of IL-17RA promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we showed that T cell-intrinsic IL-17RA signaling is necessary to promote the MHV68-driven germinal center response by supporting CD4+ T follicular helper cell expansion. We also found that T cell-intrinsic IL-17RA signaling contributes to but is not solely responsible for the systemic proviral role of IL-17RA signaling, highlighting the multifaceted function of IL-17RA signaling during MHV68 infection.


Assuntos
Infecções por Vírus Epstein-Barr , Gammaherpesvirinae , Infecções por Herpesviridae , Linfoma de Células B , Rhadinovirus , Animais , Herpesvirus Humano 4 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-17 , Linfócitos T/metabolismo , Latência Viral
3.
J Virol ; 96(10): e0002722, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35481781

RESUMO

Noncanonical NF-κB signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin ß receptor (LTßR), and B-cell-activating factor receptor (BAFF-R). The noncanonical pathway is required at multiple stages of B cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt noncanonical NF-κB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IκB kinase α (IKKα), named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NF-κB-inducing kinase (NIK). We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTßR-mediated activation of NF-κB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in the colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (dpi). Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the noncanonical NF-κB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCE The latency programs of the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block noncanonical NF-κB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKα that is nonresponsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the noncanonical NF-κB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.


Assuntos
Infecções por Herpesviridae , Quinase I-kappa B , NF-kappa B , Rhadinovirus , Latência Viral , Animais , Infecções por Herpesviridae/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Rhadinovirus/fisiologia , Transdução de Sinais , Latência Viral/genética
4.
Viruses ; 14(4)2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35458443

RESUMO

Equid herpesvirus 2 (EHV-2) and 5 (EHV-5) are two γ-herpesviruses that are commonly detected from horses worldwide, based on several cross-sectional molecular surveys. Comparatively few studies examined the dynamics of γ-herpesvirus infection over time in a group of horses. The aim of the current study was to investigate the dynamics of EHV-2/5 infections among mares and their foals at three Polish national studs with different breeds of horses: Arabians, Thoroughbreds and Polish Konik horses. Nasal swabs were collected from each of 38 mare-foal pairs monthly for a period of 6 to 8 months. Virus-specific quantitative PCR assays were used to determine the viral load of EHV-2 and EHV-5 in each sample. All 76 horses sampled were positive for EHV-2 or EHV-5 on at least one sampling occasion. The majority (73/76, 96%) were infected with both EHV-2 and EHV-5. In general, the mean load of viral DNA was higher in samples from foals than from mares, but similar for EHV-2 and EHV-5 at most sampling occasions. There was, however, a considerable variability in the viral DNA load between samples collected at different times from the same foal, as well as between samples from different foals. The latter was more apparent for EHV-2 than for EHV-5. All foals became infected with both viruses early in life, before weaning, and remained positive on all, or most, subsequent samplings. The virus shedding by mares was more intermittent, indicating the existence of age-related differences. Overall, the data presented extend our knowledge of EHV-2/5 epidemiology among mares and foals.


Assuntos
Infecções por Herpesviridae , Herpesvirus Equídeo 1 , Herpesvirus Equídeo 4 , Doenças dos Cavalos , Rhadinovirus , Animais , Estudos Transversais , DNA Viral/genética , Feminino , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 4/genética , Cavalos , Cinética , Polônia/epidemiologia , Rhadinovirus/genética
5.
Viruses ; 14(1)2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-35062301

RESUMO

Human respiratory syncytial virus (hRSV) infection brings a wide spectrum of clinical outcomes, from a mild cold to severe bronchiolitis or even acute interstitial pneumonia. Among the known factors influencing this clinical diversity, genetic background has often been mentioned. In parallel, recent evidence has also pointed out that an early infectious experience affects heterologous infections severity. Here, we analyzed the importance of these two host-related factors in shaping the immune response in pneumoviral disease. We show that a prior gammaherpesvirus infection improves, in a genetic background-dependent manner, the immune system response against a subsequent lethal dose of pneumovirus primary infection notably by inducing a systematic expansion of the CD8+ bystander cell pool and by modifying the resident alveolar macrophages (AMs) phenotype to induce immediate cyto/chemokinic responses upon pneumovirus exposure, thereby drastically attenuating the host inflammatory response without affecting viral replication. Moreover, we show that these AMs present similar rapid and increased production of neutrophil chemokines both in front of pneumoviral or bacterial challenge, confirming recent studies attributing a critical antibacterial role of primed AMs. These results corroborate other recent studies suggesting that the innate immunity cells are themselves capable of memory, a capacity hitherto reserved for acquired immunity.


Assuntos
Patrimônio Genético , Infecções por Herpesviridae/imunologia , Macrófagos Alveolares/imunologia , Infecções por Pneumovirus/imunologia , Pneumovirus/imunologia , Rhadinovirus/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Feminino , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Imunidade Inata , Inflamação/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Infecções Pneumocócicas/imunologia , Pneumovirus/fisiologia , Infecções por Pneumovirus/genética , Infecções por Pneumovirus/patologia , Infecções por Pneumovirus/virologia , Rhadinovirus/fisiologia
6.
mBio ; 12(6): e0211321, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34933450

RESUMO

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens and may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action is well understood. We report here that in some cells but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.


Assuntos
Antígenos de Diferenciação/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 8/fisiologia , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/imunologia , Rhadinovirus/fisiologia , Animais , Antígenos de Diferenciação/genética , Coinfecção/genética , Coinfecção/imunologia , Coinfecção/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Rhadinovirus/genética , Especificidade da Espécie , Internalização do Vírus , Replicação Viral
7.
J Virol ; 95(23): e0155521, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34523965

RESUMO

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.


Assuntos
Herpesviridae/genética , Herpesviridae/fisiologia , Recombinação Genética , Internalização do Vírus , Animais , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Nariz , Mucosa Olfatória/patologia , Fases de Leitura Aberta/genética , Receptores Odorantes , Rhadinovirus/genética
8.
Annu Rev Virol ; 8(1): 349-371, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586873

RESUMO

Gammaherpesviruses are an important class of oncogenic pathogens that are exquisitely evolved to their respective hosts. As such, the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) do not naturally infect nonhuman primates or rodents. There is a clear need to fully explore mechanisms of gammaherpesvirus pathogenesis, host control, and immune evasion in the host. A gammaherpesvirus pathogen isolated from murid rodents was first reported in 1980; 40 years later, murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68) infection of laboratory mice is a well-established pathogenesis system recognized for its utility in applying state-of-the-art approaches to investigate virus-host interactions ranging from the whole host to the individual cell. Here, we highlight recent advancements in our understanding of the processes by which MHV68 colonizes the host and drives disease. Lessons that inform KSHV and EBV pathogenesis and provide future avenues for novel interventions against infection and virus-associated cancers are emphasized.


Assuntos
Infecções por Vírus Epstein-Barr , Gammaherpesvirinae , Infecções por Herpesviridae , Rhadinovirus , Animais , Herpesvirus Humano 4 , Camundongos , Latência Viral
9.
Vector Borne Zoonotic Dis ; 21(10): 822-826, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339313

RESUMO

Ecology and epidemiology of murine gammaherpesvirus 68 (MHV-68) have been intensively studied since the isolation of the virus from murid rodents in 1976. This virus was detected in various mammalian species that share the biotope with rodent reservoirs of MHV-68. However, a survey of MHV-68 in birds has not so far been performed. Therefore, the aim of this study was to investigate the presence of MHV-68 in blood samples from two bird species captured at four localities in Slovakia. Using the nested PCR targeting ORF50 gene of MHV-68, we confirmed the presence of MHV-68 DNA in 9 out of 57 blood samples from Great tits (Parus major) (prevalence 15.8%, confidence interval [95% CI]: 8.5-27.4) and in 3 out of 43 blood samples from Eurasian blue tits (Cyanistes caeruleus) (prevalence 7.0%, 95% CI: 2.4-18.6). Our results suggest that not only mammals but also birds may serve as reservoirs for MHV-68, providing further evidence that MHV-68 is capable of frequent cross-species transmission.


Assuntos
Rhadinovirus , Animais , Aves , Camundongos , Reação em Cadeia da Polimerase/veterinária , Rhadinovirus/genética , Roedores , Eslováquia
10.
Viruses ; 13(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073189

RESUMO

Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.


Assuntos
Sistemas CRISPR-Cas , DNA Polimerase Dirigida por DNA/genética , Edição de Genes , Genes Virais , Mutação , Rhadinovirus/fisiologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Códon , DNA Polimerase Dirigida por DNA/química , Aptidão Genética , Genótipo , Humanos , Camundongos , Modelos Moleculares , Fenótipo , Conformação Proteica , Rhadinovirus/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Front Cell Infect Microbiol ; 11: 648055, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33898329

RESUMO

Viruses are known for their ability to alter host gene expression. Kaposi sarcoma-associated herpesvirus has two proteins that obstruct host gene expression. KSHV SOX, encoded by the open reading frame 37 (ORF37), induces a widespread cytoplasmic mRNA degradation and a block on mRNA nuclear export. The other KSHV protein, encoded by the open reading frame 10 (ORF10), was recently identified to inhibit host gene expression through its direct function on the cellular mRNA export pathway. In this review, we summarize the studies on both SOX and ORF10 in efforts to elucidate their mechanisms. We also discuss how the findings based on a closely related rodent virus, murine gammaherpesvirus-68 (MHV-68), complement the KSHV findings to decipher the role of these two proteins in viral pathogenesis.


Assuntos
Herpesvirus Humano 8 , Rhadinovirus , Transporte Ativo do Núcleo Celular , Animais , Expressão Gênica , Herpesvirus Humano 8/genética , Camundongos , Estabilidade de RNA , Replicação Viral
12.
J Virol ; 95(14): e0033021, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33910957

RESUMO

A prophylactic vaccine that confers durable protection against human immunodeficiency virus (HIV) would provide a valuable tool to prevent new HIV/AIDS cases. As herpesviruses establish lifelong infections that remain largely subclinical, the use of persistent herpesvirus vectors to deliver HIV antigens may facilitate the induction of long-term anti-HIV immunity. We previously developed recombinant (r) forms of the gamma-herpesvirus rhesus monkey rhadinovirus (rRRV) expressing a replication-incompetent, near-full-length simian immunodeficiency virus (SIVnfl) genome. We recently showed that 8/16 rhesus macaques (RMs) vaccinated with a rDNA/rRRV-SIVnfl regimen were significantly protected against intrarectal (i.r.) challenge with SIVmac239. Here we investigated the longevity of this vaccine-mediated protection. Despite receiving no additional booster immunizations, the protected rDNA/rRRV-SIVnfl vaccinees maintained detectable cellular and humoral anti-SIV immune responses for more than 1.5 years after the rRRV boost. To assess if these responses were still protective, the rDNA/rRRV-SIVnfl vaccinees were subjected to a second round of marginal-dose i.r. SIVmac239 challenges, with eight SIV-naive RMs serving as concurrent controls. After three SIV exposures, 8/8 control animals became infected, compared to 3/8 vaccinees. This difference in SIV acquisition was statistically significant (P = 0.0035). The three vaccinated monkeys that became infected exhibited significantly lower viral loads than those in unvaccinated controls. Collectively, these data illustrate the ability of rDNA/rRRV-SIVnfl vaccination to provide long-term immunity against stringent mucosal challenges with SIVmac239. Future work is needed to identify the critical components of this vaccine-mediated protection and the extent to which it can tolerate sequence mismatches in the challenge virus. IMPORTANCE We report on the long-term follow-up of a group of rhesus macaques (RMs) that received an AIDS vaccine regimen and were subsequently protected against rectal acquisition of simian immunodeficiency virus (SIV) infection. The vaccination regimen employed included a live recombinant herpesvirus vector that establishes persistent infection in RMs. Consistent with the recurrent SIV antigen expression afforded by this herpesvirus vector, vaccinees maintained detectable SIV-specific immune responses for more than 1.5 years after the last vaccination. Importantly, these vaccinated RMs were significantly protected against a second round of rectal SIV exposures performed 1 year after the first SIV challenge phase. These results are relevant for HIV vaccine development because they show the potential of herpesvirus-based vectors to maintain functional antiretroviral immunity without the need for repeated boosting.


Assuntos
Vetores Genéticos , Rhadinovirus/genética , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genética , Animais , Anticorpos Antivirais/imunologia , Feminino , Seguimentos , Imunogenicidade da Vacina , Memória Imunológica , Macaca mulatta , Masculino , Rhadinovirus/imunologia , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Fatores de Tempo
13.
PLoS Pathog ; 17(3): e1008979, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657166

RESUMO

The rhesus monkey rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposi's sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by ~60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility and cell-cell fusion of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 and reduced RRV infection in a cell type-specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV.


Assuntos
Macaca mulatta/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Rhadinovirus/patogenicidade , Animais , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/genética , Humanos , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus
14.
Ann Clin Transl Neurol ; 8(2): 456-470, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33440071

RESUMO

OBJECTIVE: To determine whether animals with Japanese macaque encephalomyelitis (JME), a spontaneous demyelinating disease similar to multiple sclerosis (MS), harbor myelin-specific T cells in their central nervous system (CNS) and periphery. METHODS: Mononuclear cells (MNCs) from CNS lesions, cervical lymph nodes (LNs) and peripheral blood of Japanese macaques (JMs) with JME, and cervical LN and blood MNCs from healthy controls or animals with non-JME conditions were analyzed for the presence of myelin-specific T cells and changes in interleukin 17 (IL-17) and interferon gamma (IFNγ) expression. RESULTS: Demyelinating JME lesions contained CD4+ T cells and CD8+ T cells specific to myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and/or proteolipid protein (PLP). CD8+ T-cell responses were absent in JME peripheral blood, and in age- and sex-matched controls. However, CD4+ Th1 and Th17 responses were detected in JME peripheral blood versus controls. Cervical LN MNCs from eight of nine JME animals had CD3+ T cells specific for MOG, MBP, and PLP that were not detected in controls. Mapping myelin epitopes revealed a heterogeneity in responses among JME animals. Comparison of myelin antigen sequences with those of JM rhadinovirus (JMRV), which is found in JME lesions, identified six viral open reading frames (ORFs) with similarities to myelin antigen sequences. Overlapping peptides to these JMRV ORFs did not induce IFNγ responses. INTERPRETATIONS: JME possesses an immune-mediated component that involves both CD4+ and CD8+ T cells specific for myelin antigens. JME may shed new light on inflammatory demyelinating disease pathogenesis linked to gamma-herpesvirus infection.


Assuntos
Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/patologia , Encefalomielite/diagnóstico por imagem , Encefalomielite/patologia , Bainha de Mielina/imunologia , Linfócitos T/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Desmielinizantes/virologia , Encefalomielite/virologia , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Feminino , Infecções por Herpesviridae/imunologia , Interferon gama/análise , Interleucina-17/análise , Macaca fuscata , Masculino , Doenças dos Macacos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/imunologia , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/imunologia , Bainha de Mielina/patologia , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/imunologia , Rhadinovirus/genética , Rhadinovirus/imunologia
15.
JCI Insight ; 6(2)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33491663

RESUMO

The aryl-hydrocarbon receptor (AHR) is an intracellular sensor of aromatic hydrocarbons that sits at the top of various immunomodulatory pathways. Here, we present evidence that AHR plays a role in controlling IL-17 responses and the development of pulmonary fibrosis in response to respiratory pathogens following bone marrow transplant (BMT). Mice infected intranasally with gamma-herpesvirus 68 (γHV-68) following BMT displayed elevated levels of the AHR ligand, kynurenine (kyn), in comparison with control mice. Inhibition or genetic ablation of AHR signaling resulted in a significant decrease in IL-17 expression as well as a reduction in lung pathology. Lung CD103+ DCs expressed AHR following BMT, and treatment of induced CD103+ DCs with kyn resulted in altered cytokine production in response to γHV-68. Interestingly, mice deficient in the kyn-producing enzyme indolamine 2-3 dioxygenase showed no differences in cytokine responses to γHV-68 following BMT; however, isolated pulmonary fibroblasts infected with γHV-68 expressed the kyn-producing enzyme tryptophan dioxygenase (TDO2). Our data indicate that alterations in the production of AHR ligands in response to respiratory pathogens following BMT results in a pro-Th17 phenotype that drives lung pathology. We have further identified the TDO2/AHR axis as a potentially novel form of intercellular communication between fibroblasts and DCs that shapes immune responses to respiratory pathogens.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transplante de Medula Óssea/efeitos adversos , Fibrose Pulmonar/etiologia , Receptores de Hidrocarboneto Arílico/metabolismo , Rhadinovirus/patogenicidade , Triptofano Oxigenase/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Indolamina-Pirrol 2,3,-Dioxigenase/deficiência , Interleucina-17/biossíntese , Cinurenina/metabolismo , Ligantes , Pulmão/imunologia , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Rhadinovirus/imunologia , Transdução de Sinais , Células Th17/imunologia
17.
J Equine Vet Sci ; 94: 103225, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33077072

RESUMO

Idiopathic systemic granulomatous disease (ISGD), also known as equine sarcoidosis is an uncommon disease of horses, manifesting in exfoliative dermatitis and granulomatous inflammation in various organs. The current report presents a case of a 15-year-old Hanoverian mare with a 4-month history of weight loss, recurrent fever, skin lesions, and movement disorders. Pathological examination revealed granulomatous and necrotizing inflammation in the skin, regional lymph nodes, and cerebellum. Based on histological, immunohistochemical, and microbiological findings, the diagnosis of ISGD was made. Sequencing of the polymerase chain reaction product of pooled brain tissue revealed the presence of equid gammaherpesvirus 2 DNA. This case is the first description of generalized ISGD with granulomatous dermatitis simultaneously affecting the skin and cerebellum.


Assuntos
Doenças dos Cavalos , Rhadinovirus , Dermatopatias , Animais , Cerebelo , Feminino , Granuloma/veterinária , Cavalos , Dermatopatias/veterinária
18.
J Virol ; 95(1)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33028711

RESUMO

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/imunologia , Infecções por Herpesviridae/virologia , Rhadinovirus/fisiologia , Proteínas Virais/imunologia , Ativação Viral , Animais , Antígenos CD19/metabolismo , Linfócitos B/virologia , Diferenciação Celular , Proliferação de Células , Centro Germinativo/imunologia , Centro Germinativo/virologia , Infecções por Herpesviridae/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Camundongos , Rhadinovirus/genética , Rhadinovirus/metabolismo , Proteínas Virais/genética , Latência Viral
19.
Antiviral Res ; 182: 104901, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32763314

RESUMO

Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described specific changes in MHV-68 protein kinase (PK) or thymidine kinase (TK) associated with resistance to various purine or pyrimidine nucleoside analogues, respectively. To investigate how specific TK and PK mutations affect viral replication capacity, we performed dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals in Vero cells. The composition of the mixed viral population was analyzed using next-generation sequencing and relative fitness of seven MHV-68 PK or TK mutants was calculated based on the frequency of viral variants at the time of infection and after 5-days growth. A MHV-68 mutant losing the PK function due to a 2-nucleotide deletion was less fit than the wild-type virus in absence of antivirals, consistent with the essential role of viral PKs during lytic replication, but overgrew the wild-type virus under pressure of purine nucleosides. TK mutant viruses, with frameshift or missense mutations, grew equal to wild-type virus in absence of antivirals, in accordance with the viral TK function only being essential in non-replicating or in TK-deficient cells, but were more fit when treated with pyrimidine nucleosides. Moreover, TK missense mutant viruses also increased fitness under pressure of antivirals other than pyrimidine nucleosides, indicating that MHV-68 TK mutations might influence viral fitness by acting on cellular and/or viral functions that are unrelated to nucleoside activation.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Aptidão Genética , Mutação de Sentido Incorreto , Proteínas Quinases/genética , Rhadinovirus/efeitos dos fármacos , Timidina Quinase/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Camundongos , Células NIH 3T3 , Rhadinovirus/genética , Rhadinovirus/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
20.
PLoS Pathog ; 16(7): e1008701, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735617

RESUMO

Gammaherpesviruses have evolved various strategies to take advantage of host cellular factors or signaling pathways to establish a lifelong latent infection. Like the human gammaherpesvirus Epstein-Barr virus, murine gammaherpesvirus 68 (MHV68) establishes and maintains latency in the memory B cells during infection of laboratory mice. We have previously shown that MHV68 can immortalize fetal liver-derived B cells that induce lymphomas when injected into immunodeficient mice. Here we identify interleukin 16 (IL16) as a most abundantly expressed cytokine in MHV68-immortalized B cells and show that MHV68 infection elevates IL16 expression. IL16 is not important for MHV68 lytic infection but plays a critical role in MHV68 reactivation from latency. IL16 deficiency increases MHV68 lytic gene expression in MHV68-immortalized B cells and enhances reactivation from splenic latency. Correlatively, IL16 deficiency increases the frequency of MHV68-infected plasma cells that can be attributed to enhanced MHV68 reactivation. Furthermore, similar to TPA-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus, IL16 deficiency markedly induces Tyr705 STAT3 de-phosphorylation and elevates p21 expression, which can be counteracted by the tyrosine phosphatase inhibitor orthovanadate. Importantly, orthovanadate strongly blocks MHV68 lytic gene expression mediated by IL16 deficiency. These data demonstrate that virus-induced IL16 does not directly participate in MHV68 lytic replication, but rather inhibits virus reactivation to facilitate latent infection, in part through the STAT3-p21 axis.


Assuntos
Infecções por Herpesviridae/metabolismo , Interleucina-16/metabolismo , Infecções Tumorais por Vírus/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Linfócitos B/virologia , Infecções por Herpesviridae/imunologia , Interleucina-16/imunologia , Linfoma/virologia , Camundongos , Rhadinovirus/imunologia , Rhadinovirus/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...