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1.
Nature ; 589(7842): 456-461, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33328639

RESUMO

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.


Assuntos
Autofagia/imunologia , Nexinas de Classificação/metabolismo , Vírus/imunologia , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/metabolismo , Feminino , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Nexinas de Classificação/deficiência , Nexinas de Classificação/genética , Proteínas de Transporte Vesicular/metabolismo
2.
J Virol ; 98(5): e0004724, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38651898

RESUMO

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.


Assuntos
Norovirus , Tropismo Viral , Internalização do Vírus , Animais , Humanos , Camundongos , Infecções por Caliciviridae/virologia , Genoma Viral , Células HeLa , Especificidade de Hospedeiro , Mutação , Norovirus/genética , Norovirus/fisiologia , Receptores Virais/metabolismo , Receptores Virais/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Ligação Viral , Replicação Viral
3.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34282021

RESUMO

RNA viruses exist as genetically heterogeneous populations due to high mutation rates, and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach-selecting for the earliest viral progeny-could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles.


Assuntos
Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Replicação Viral/genética , Animais , Clonagem Molecular , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mutação , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Replicação Viral/fisiologia
4.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852778

RESUMO

Enteric viruses infect the gastrointestinal tract, and bacteria can promote replication and transmission of several enteric viruses. Viruses can be inactivated by exposure to heat or bleach, but poliovirus, coxsackievirus B3, and reovirus can be stabilized by bacteria or bacterial polysaccharides, limiting inactivation and aiding transmission. We previously demonstrated that certain N-acetylglucosamine (GlcNAc)-containing polysaccharides can stabilize poliovirus. However, the detailed virus-glycan binding specificity and glycan chain length requirements, and thus the mechanism of virion stabilization, have been unclear. A previous limitation was our lack of defined-length glycans to probe mechanisms and consequences of virus-glycan interactions. Here, we generated a panel of polysaccharides and oligosaccharides to determine the properties required for binding and stabilization of poliovirus. Poliovirus virions are nonenveloped icosahedral 30-nm particles with 60 copies of each of four capsid proteins, VP1 to VP4. VP1 surrounds the 5-fold axis, and our past work indicates that this region likely contains the glycan binding site. We found that relatively short GlcNAc oligosaccharides, such as a six-unit GlcNAc oligomer, can bind poliovirus but fail to enhance virion stability. Virion stabilization required binding of long GlcNAc polymers of greater than 20 units. Our data suggest a model where GlcNAc polymers of greater than 20 units bind and bridge adjacent 5-fold axes, thus aiding capsid rigidity and stability. This study provides a deeper understanding of enteric virus-bacterial glycan interactions, which are important for virion environmental stability and transmission.IMPORTANCE Enteric viruses are transmitted through the fecal-oral route, but how enteric viruses survive in the environment is unclear. Previously, we found that bacterial polysaccharides enhance poliovirus stability against heat or bleach inactivation, but the specific molecular requirements have been unknown. Here, we showed that certain short-chain oligosaccharides can bind to poliovirus but do not increase virion stability. Long-chain polysaccharides bind and may bridge adjacent sites on the viral surface, thus increasing capsid rigidity and stability. This work defines the unique interactions of poliovirus and glycans, which provides insight into virion environmental stability and transmission.


Assuntos
Enterovirus/metabolismo , Oligossacarídeos/metabolismo , Poliovirus/fisiologia , Polissacarídeos , Vírion/fisiologia , Animais , Bactérias/metabolismo , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiops , Infecções por Enterovirus/virologia , Células HeLa , Humanos , Lipopolissacarídeos/metabolismo , Células Vero
5.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666382

RESUMO

The gastrointestinal tract presents a formidable barrier for pathogens to initiate infection. Despite this barrier, enteroviruses, including coxsackievirus B3 (CVB3), successfully penetrate the intestine to initiate infection and spread systemically prior to shedding in stool. However, the effect of the gastrointestinal barrier on CVB3 population dynamics is relatively unexplored, and the selective pressures acting on CVB3 in the intestine are not well characterized. To examine viral population dynamics in orally infected mice, we produced over 100 CVB3 clones harboring nine unique nucleotide "barcodes." Using this collection of barcoded viruses, we found diverse viral populations throughout each mouse within the first day postinfection, but by 48 h the viral populations were dominated by fewer than three barcoded viruses in intestinal and extraintestinal tissues. Using light-sensitive viruses to track replication status, we found that diverse viruses had replicated prior to loss of diversity. Sequencing whole viral genomes from samples later in infection did not reveal detectable viral adaptations. Surprisingly, orally inoculated CVB3 was detectable in pancreas and liver as soon as 20 min postinoculation, indicating rapid systemic dissemination. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues. These results underscore a complex dynamic between dissemination and clearance for an enteric virus.IMPORTANCE Enteric viruses initiate infection in the gastrointestinal tract but can disseminate to systemic sites. However, the dynamics of viral dissemination are unclear. In this study, we created a library of 135 barcoded coxsackieviruses to examine viral population diversity across time and space following oral inoculation of mice. Overall, we found that the broad population of viruses disseminates early, followed by monopolization of mouse tissues with three or fewer pool members at later time points. Interestingly, we detected virus in systemic tissues such as pancreas and liver just 20 min after oral inoculation. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues.


Assuntos
Código de Barras de DNA Taxonômico , Enterovirus Humano B/fisiologia , Infecções por Enterovirus , Replicação Viral , Animais , Infecções por Enterovirus/genética , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/patologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout
6.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30567995

RESUMO

Enteric viruses, including poliovirus, are spread by the fecal-oral route. In order to persist and transmit to a new host, enteric virus particles must remain stable once they are in the environment. Environmental stressors such as heat and disinfectants can inactivate virus particles and prevent viral transmission. It has been previously demonstrated that bacteria or bacterial surface glycans can enhance poliovirus virion stability and limit inactivation from heat or bleach. While investigating the mechanisms underlying bacterially enhanced virion thermal stability, we identified and characterized a poliovirus (PV) mutant with increased resistance to heat inactivation. The M132V mutant harbors a single amino acid change in the VP1 capsid coding that is sufficient to confer heat resistance but not bleach resistance. Although the M132V virus was stable in the absence of bacteria or feces at most temperatures, M132V virus was stabilized by feces at very high temperatures. M132V PV had reduced specific infectivity and RNA uncoating compared with those of wild-type (WT) PV, but viral yields in HeLa cells were similar. In orally inoculated mice, M132V had a slight fitness cost since fecal titers were lower and 12.5% of fecal viruses reverted to the WT. Overall, this work sheds light on factors that influence virion stability and fitness.IMPORTANCE Viruses spread by the fecal-oral route need to maintain viability in the environment to ensure transmission. Previous work indicated that bacteria and bacterial surface polysaccharides can stabilize viral particles and enhance transmission. To explore factors that influence viral particle stability, we isolated a mutant poliovirus that is heat resistant. This mutant virus does not require feces for stability at most temperatures but can be stabilized by feces at very high temperatures. Even though the mutant virus is heat resistant, it is susceptible to inactivation by treatment with bleach. This work provides insight into how viral particles maintain infectivity in the environment.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/fisiologia , Mutação/genética , Poliovirus/genética , Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HeLa , Temperatura Alta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Poliomielite/virologia , RNA Viral/genética , Vírion/genética
7.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31511379

RESUMO

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota.IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota.


Assuntos
Infecções por Enterovirus/microbiologia , Infecções por Enterovirus/virologia , Enterovirus/efeitos dos fármacos , Enterovirus/patogenicidade , Microbiota/efeitos dos fármacos , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Bactérias/classificação , Infecções por Coxsackievirus , Modelos Animais de Doenças , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Picornaviridae/efeitos dos fármacos , Picornaviridae/patogenicidade , Poliovirus/efeitos dos fármacos , Poliovirus/patogenicidade , Vancomicina/farmacologia , Replicação Viral/efeitos dos fármacos
8.
J Virol ; 91(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28100612

RESUMO

Coxsackievirus is an enteric virus that initiates infection in the gastrointestinal tract before disseminating to peripheral tissues to cause disease, but intestinal factors that influence viral replication are understudied. Furthermore, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. While mouse models mimicking human pathogenesis have been well characterized, many of these experiments use intraperitoneal injection of coxsackievirus to infect mice, bypassing the intestine. In light of recent studies identifying intestinal factors, such as the microbiota, that alter enteric viral replication, we sought to investigate coxsackievirus replication within the intestine. Here, we orally infected mice with coxsackievirus B3 (CVB3) and found that CVB3 replication in the intestine is sex dependent. CVB3 replicated efficiently in the intestine of male mice but not female mice. Additionally, we found that the type I interferon response and sex hormones can alter both viral replication and lethality. Overall, these data suggest that sex and the immune response play a vital role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.IMPORTANCE Sex bias in severe sequelae from enteric viral infections has been observed. Since viruses have evolved to achieve optimal levels of fitness in their environmental niches, it is imperative to study viruses at the site of initial replication. Here, we used an oral inoculation system for CVB3, which follows the natural route of infection in the gastrointestinal tract. We found that sex can influence the replication of CVB3 in the intestine. Additionally, the type I interferon response and sex hormones alter both CVB3 intestinal replication and lethality. Overall this work highlights the fact that sex should be considered in investigations of enteric viral replication and pathogenesis.


Assuntos
Infecções por Enterovirus/virologia , Enterovirus/fisiologia , Intestinos/virologia , Replicação Viral , Animais , Infecções por Enterovirus/imunologia , Feminino , Intestinos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Caracteres Sexuais
9.
J Gen Virol ; 96(Pt 6): 1328-1339, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25646269

RESUMO

The host and viral factors that influence disease outcome during flavivirus infections are not fully understood. Using the live attenuated yellow fever virus (YFV) vaccine strain 17D as a model system we evaluated how viral dose, inoculation route and immunopathogenesis contributed to disease outcome in mice deficient in the type I IFN response. We found that YFV-17D infection of IFN-α/ß receptor knockout mice resulted in three distinct disease outcomes: no clinical signs of disease, fatal viscerotropic disease or fatal neurotropic disease. Interestingly, viral load at disease onset did not correlate with disease outcome. However, we found increased immune infiltrates in the brain tissues of mice that developed neurotropic disease. Additionally, mice that developed viscerotropic disease, as characterized by liver and spleen pathology and/or intestinal haemorrhage, had significantly elevated levels of alanine aminotransferase, monocyte chemotactic protein and IFN-inducible protein (IP)-10 as compared with mice with no clinical signs of disease or neurotropic disease. Furthermore, mice treated with recombinant IP-10 throughout YFV-17D infection showed increased mortality and an increased percentage of mice with viscerotropic disease. Our results demonstrated that viral load did not correlate with pathogenesis, and the host immune response played a pivotal role in disease outcome and contributed to YFV-17D pathogenesis in mice.


Assuntos
Modelos Animais de Doenças , Febre Amarela/patologia , Febre Amarela/virologia , Vírus da Febre Amarela/fisiologia , Alanina Transaminase/sangue , Animais , Encéfalo/patologia , Quimiocina CCL2/sangue , Quimiocina CXCL10/sangue , Hemorragia Gastrointestinal , Interferon Tipo I/deficiência , Intestinos/patologia , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/patologia , Análise de Sobrevida , Carga Viral
11.
J Virol ; 87(22): 12392-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24027319

RESUMO

Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod vectors and vertebrate hosts. While barriers limiting arbovirus population diversity have been observed in mosquitoes, whether barriers exist in vertebrate hosts is unclear. To investigate whether arboviruses encounter bottlenecks during dissemination in the vertebrate host, we infected immunocompetent mice and immune-deficient mice lacking alpha/beta interferon (IFN-α/ß) receptors (IFNAR⁻/⁻ mice) with a pool of genetically marked viruses to evaluate dissemination and host barriers. We used the live attenuated vaccine strain YFV-17D, which contains many mutations compared with virulent YFV. We found that intramuscularly injected immunocompetent mice did not develop disease and that viral dissemination was restricted. Conversely, 32% of intramuscularly injected IFNAR⁻/⁻ mice developed disease. By following the genetically marked viruses over time, we found broad dissemination in IFNAR⁻/⁻ mice followed by clearance. The patterns of viral dissemination were similar in mice that developed disease and mice that did not develop disease. Unlike our previous results with poliovirus, these results suggest that YFV-17D encounters no major barriers during dissemination within a vertebrate host in the absence of the type I IFN response.


Assuntos
Camundongos Knockout/virologia , Receptor de Interferon alfa e beta/fisiologia , Viremia/transmissão , Replicação Viral , Febre Amarela/virologia , Vírus da Febre Amarela/patogenicidade , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mutação/genética , Taxa de Sobrevida , Carga Viral , Viremia/virologia , Febre Amarela/genética , Febre Amarela/mortalidade , Vírus da Febre Amarela/genética
13.
Cell Host Microbe ; 32(9): 1457-1459, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39265530

RESUMO

Breastfeeding provides infection protection for several pathogens but not for noroviruses. Mechanisms explaining this discrepancy have been unclear. In this issue of Cell Host & Microbe, Peiper et al. demonstrate that while breastmilk protects mice from intestinal damage, it promotes neonatal murine norovirus infection due to maternal-derived bile acids.1.


Assuntos
Ácidos e Sais Biliares , Infecções por Caliciviridae , Leite Humano , Norovirus , Animais , Ácidos e Sais Biliares/metabolismo , Infecções por Caliciviridae/virologia , Camundongos , Leite Humano/virologia , Leite Humano/química , Humanos , Feminino , Aleitamento Materno , Gastroenterite/virologia
14.
bioRxiv ; 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38260699

RESUMO

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select for mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. While viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when entry was bypassed, suggesting the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in mouse BV2 cells. Although the mutant viruses had increased fitness in HeLa cells, they did not have increased fitness in mice. Overall, this work suggests that MNV tropism is not only determined by the presence of the viral receptor but also post-entry factors.

15.
J Clin Invest ; 134(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37917202

RESUMO

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.


Assuntos
Agregados Proteicos , Vírus , Camundongos , Animais , Autofagia/genética , Fosforilação , Camundongos Knockout , Proteínas do Capsídeo , Antígenos Virais , Proteínas Serina-Treonina Quinases/genética
16.
J Virol ; 85(14): 7273-83, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21543469

RESUMO

Many individuals infected with hepatitis C virus (HCV) develop a chronic infection, and of those who are treated with pegylated interferon and ribavirin (RBV), many do not respond. While the nucleoside analog RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV's mechanism is controversial. Most of RBV's proposed mechanisms require RBV import into cells. Therefore, we explored whether host-based RBV resistance develops through reduced cellular uptake, akin to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on HCV replication in cultured hepatoma Huh7.5 liver cells and whether RBV resistance develops in HCV patients. When Huh7.5 cells were exposed to RBV, resistance developed through reduced RBV uptake via the ENT1 nucleoside transporter and antiviral efficacy was reduced. The uptake defect in RBV-resistant cells was specific to RBV, since transport of another ENT1 substrate, cytidine, was unaffected. Importantly, RBV uptake significantly declined in HCV patient peripheral blood mononuclear cells (PBMCs) following 4 weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance and suggest that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Ribavirina/farmacologia , Linhagem Celular Tumoral , Farmacorresistência Viral , Hepacivirus/fisiologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Virol ; 85(19): 9778-88, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21795346

RESUMO

The propensity of RNA viruses to revert attenuating mutations contributes to disease and complicates vaccine development. Despite the presence of virulent revertant viruses in some live-attenuated vaccines, disease from vaccination is rare. This suggests that in mixed viral populations, attenuated viruses may limit the pathogenesis of virulent viruses, thus establishing a virulence threshold. Here we examined virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that a 1,000-fold excess of the attenuated Sabin strain of poliovirus was protective against disease induced by the virulent Mahoney strain. Protection was induced locally, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting that one mechanism controlling virulence thresholds may be competition for a viral receptor. Additionally, the type I interferon response reduces poliovirus pathogenesis; therefore, we examined virulence thresholds in mice lacking the alpha/beta interferon receptor. We found that the attenuated virus was virulent in immunodeficient mice due to the enhanced replication and reversion of attenuating mutations. Therefore, while the type I interferon response limits the virulence of the attenuated strain by reducing replication, protection from disease conferred by the attenuated strain in immunocompetent mice can occur independently of replication. Our results identified mechanisms controlling the virulence of mixed viral populations and indicate that live-attenuated vaccines containing virulent virus may be safe, as long as virulent viruses are present at levels below a critical threshold.


Assuntos
Poliomielite/virologia , Poliovirus/patogenicidade , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças dos Roedores/virologia , Virulência , Ligação Viral
18.
PLoS Pathog ; 6(3): e1000791, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20221252

RESUMO

Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle-sciatic nerve-spinal cord-brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) alpha/beta receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN alpha/beta receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses.


Assuntos
Transporte Axonal/imunologia , Interferon Tipo I/metabolismo , Poliomielite/imunologia , Poliovirus/imunologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Células HeLa , Humanos , Injeções Intramusculares , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/lesões , Músculo Esquelético/inervação , Músculo Esquelético/virologia , Neurônios/imunologia , Neurônios/virologia , Poliomielite/fisiopatologia , Poliomielite/virologia , Poliovirus/crescimento & desenvolvimento , Poliovirus/metabolismo , Receptor de Interferon alfa e beta/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/imunologia , Nervo Isquiático/virologia , Replicação Viral/imunologia
19.
mSphere ; 7(3): e0004622, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35531660

RESUMO

Enteric viruses encounter various bacteria in the host, which can impact infection outcomes. The interactions between noroviruses and enteric bacteria are not well understood. Previous work determined that murine norovirus (MNV), a model norovirus, had decreased replication in antibiotic-treated mice compared with conventional mice. Although this suggests that the microbiota promotes MNV infection, the mechanisms are not completely understood. Additionally, prior work with other enteric viruses, such as poliovirus and coxsackievirus B3, demonstrated that virions bind bacteria, and exposure to bacteria stabilizes viral particles and limits premature RNA release. Therefore, we examined interactions between MNV and specific bacteria and the consequences of these interactions. We found that the majority of Gram-positive bacteria tested stabilized MNV, while Gram-negative bacteria did not stabilize MNV. Both Gram-positive and Gram-negative bacteria bound to MNV. However, bacterial binding alone was not sufficient for virion stabilization, since Gram-negative bacteria bound MNV but did not stabilize virions. Additionally, we found that bacteria conditioned medium also stabilized MNV and this stabilization may be due to a small heat-stable molecule. Overall, this work identifies specific bacteria and bacterial components that stabilize MNV and may impact virion stability in the environment. IMPORTANCE Enteric viruses are exposed to a wide variety of bacteria in the intestine, but the effects of bacteria on viral particles are incompletely understood. We found that murine norovirus (MNV) virion stability is enhanced in the presence of several Gram-positive bacterial strains. Virion-stabilizing activity was also present in bacterial culture medium, and activity was retained upon heat or protease treatment. These results suggest that certain bacteria and bacterial products may promote MNV stability in the environment, which could influence viral transmission.


Assuntos
Infecções por Enterovirus , Enterovirus , Norovirus , Animais , Antibacterianos , Bactérias , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Camundongos
20.
Gastroenterology ; 139(1): 154-62.e4, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20303352

RESUMO

BACKGROUND & AIMS: The therapeutic mechanisms of ribavirin for hepatitis C are unclear. Microarray analyses have shown that ribavirin increases induction of interferon-stimulated genes. We evaluated viral kinetics, serum cytokine expression, and viral mutagenesis during early stages of peginterferon therapy with and without ribavirin. METHODS: Fifty patients with chronic hepatitis C virus (HCV) infection genotype 1 were randomly assigned to groups that were given peginterferon alpha-2a, with or without ribavirin, for 4 weeks; all patients then received an additional 44 weeks of combination therapy. First- and second-phase viral kinetics were evaluated. Serum levels of interferon-gamma-inducible protein-10 (IP10), monokine induced by interferon-gamma, and monocyte chemoattractant protein 1 were quantified as measures of the interferon-stimulated genes response. NS5A and NS5B were partially sequenced, and mutation rates were calculated. RESULTS: The first-phase decrease in HCV RNA was similar between groups. Patients who received ribavirin had a more rapid second-phase decrease, compared with patients who did not receive ribavirin-particularly those with an adequate first-phase decrease (0.61 vs 0.35 log10 IU/mL/week; P = .018). At 12 hours, fold induction of serum IP10 was higher in patients given the combination therapy than those given peginterferon only (7.6- vs 3.8-fold; P = .01); however, the difference was greatest in patients with an adequate first-phase decrease in HCV RNA. IP10-induction correlated with first- and second-phase kinetics and with ribavirin serum concentrations on day 3. HCV mutation rates were similar between groups. CONCLUSIONS: Ribavirin improves the kinetics of the early response to therapy in patients with an adequate initial response to peginterferon. Induction of interferon-stimulated cytokines correlates with viral kinetics following ribavirin therapy, suggesting that ribavirin promotes interferon signaling.


Assuntos
Antivirais/farmacologia , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Ribavirina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Quimiocina CXCL10/sangue , Feminino , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Masculino , Pessoa de Meia-Idade , Mutação , Polietilenoglicóis/administração & dosagem , RNA Viral/sangue , Proteínas Recombinantes , Ribavirina/sangue , Resultado do Tratamento
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