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1.
Proc Natl Acad Sci U S A ; 116(48): 24303-24309, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719195

RESUMO

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

2.
J Exp Med ; 216(9): 2057-2070, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31270247

RESUMO

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

3.
PLoS Pathog ; 15(5): e1007798, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116799

RESUMO

Cellular antiviral programs encode molecules capable of targeting multiple steps in the virus lifecycle. Zinc-finger antiviral protein (ZAP) is a central and general regulator of antiviral activity that targets pathogen mRNA stability and translation. ZAP is diffusely cytoplasmic, but upon infection ZAP is targeted to particular cytoplasmic structures, termed stress granules (SGs). However, it remains unclear if ZAP's antiviral activity correlates with SG localization, and what molecular cues are required to induce this localization event. Here, we use Sindbis virus (SINV) as a model infection and find that ZAP's localization to SGs can be transient. Sometimes no apparent viral infection follows ZAP SG localization but ZAP SG localization always precedes accumulation of SINV non-structural protein, suggesting virus replication processes trigger SG formation and ZAP recruitment. Data from single-molecule RNA FISH corroborates this finding as the majority of cells with ZAP localization in SGs contain low levels of viral RNA. Furthermore, ZAP recruitment to SGs occurred in ZAP-expressing cells when co-cultured with cells replicating full-length SINV, but not when co-cultured with cells replicating a SINV replicon. ZAP recruitment to SGs is functionally important as a panel of alanine ZAP mutants indicate that the anti-SINV activity is correlated with ZAP's ability to localize to SGs. As ZAP is a central component of the cellular antiviral programs, these data provide further evidence that SGs are an important cytoplasmic antiviral hub. These findings provide insight into how antiviral components are regulated upon virus infection to inhibit virus spread.


Assuntos
Infecções por Alphavirus/prevenção & controle , Antivirais/farmacologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/farmacologia , Vírus Sindbis/patogenicidade , Estresse Fisiológico , Replicação Viral/efeitos dos fármacos , Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/virologia , Antivirais/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/virologia , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/virologia , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Tumorais Cultivadas
4.
J Virol ; 93(18)2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31118263

RESUMO

Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor: zinc finger antiviral protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C termini: ZAPL (long) encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously, it has been shown that ZAPL is more antiviral than ZAPS, while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP: ZAPXL (extralong) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct antiviral and/or cellular functions. By taking a gene-knockout-and-reconstitution approach, we have characterized the antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad-spectrum antiviral factor.IMPORTANCE ZAP is an IFN-induced host factor that can inhibit a wide range of viruses, and there is great interest in fully characterizing its antiviral mechanism. This is the first study that defines the antiviral capacities of individual ZAP isoforms in the absence of endogenous ZAP expression and, hence, cross talk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms: ZAPS, ZAPM, ZAPL, and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV, while all isoforms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the abilities of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.

5.
Cell Rep ; 25(6): 1385-1394.e7, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30403995

RESUMO

Zika virus (ZIKV) causes severe neurologic complications and fetal aberrations. Vaccine development is hindered by potential safety concerns due to antibody cross-reactivity with dengue virus and the possibility of disease enhancement. In contrast, passive administration of anti-ZIKV antibodies engineered to prevent enhancement may be safe and effective. Here, we report on human monoclonal antibody Z021, a potent neutralizer that recognizes an epitope on the lateral ridge of the envelope domain III (EDIII) of ZIKV and is protective against ZIKV in mice. When administered to macaques undergoing a high-dose ZIKV challenge, a single anti-EDIII antibody selected for resistant variants. Co-administration of two antibodies, Z004 and Z021, which target distinct sites on EDIII, was associated with a delay and a 3- to 4-log decrease in peak viremia. Moreover, the combination of these antibodies engineered to avoid enhancement prevented viral escape due to mutation in macaques, a natural host for ZIKV.

6.
Cell ; 172(3): 423-438.e25, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29249360

RESUMO

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.


Assuntos
Imunidade Inata , Células-Tronco Pluripotentes/imunologia , Viroses/imunologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/virologia , Especificidade da Espécie
7.
MBio ; 8(4)2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28720733

RESUMO

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.


Assuntos
Vírus da Dengue/fisiologia , Hexosiltransferases/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Linhagem Celular , Humanos
8.
Cell ; 169(4): 597-609.e11, 2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28475892

RESUMO

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Infecção por Zika virus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Brasil , Feminino , Humanos , Memória Imunológica , Leucócitos Mononucleares/imunologia , Masculino , México , Camundongos , Infecção por Zika virus/sangue
9.
PLoS Pathog ; 13(1): e1006145, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28060952

RESUMO

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.


Assuntos
Infecções por Alphavirus/prevenção & controle , Antivirais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vírus Sindbis/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Cricetinae , Células HEK293 , Humanos , Interferon Tipo I/metabolismo , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Hepatology ; 66(2): 357-370, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27642141

RESUMO

Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)-stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double-stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen-associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double-stranded to single-stranded RNA (ssRNA) correlated positively with ISG induction. In Huh-7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome-length minus strands. CONCLUSION: HCV dsRNA is the predominant form in the HCV-infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (Hepatology 2017;66:357-370).


Assuntos
Antivirais/farmacologia , Hepacivirus/genética , Hepatite C/patologia , Interferon-alfa/farmacologia , RNA de Cadeia Dupla/genética , Adulto , Biópsia por Agulha , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , RNA de Cadeia Dupla/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Valores de Referência , Sensibilidade e Especificidade
11.
J Exp Med ; 213(13): 2931-2947, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27899441

RESUMO

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2-/- mice after peripheral inoculation. Irf2-/- mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2-/- mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient µMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2-/- mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections.


Assuntos
Infecções por Alphavirus/imunologia , Linfócitos B/imunologia , Encefalopatias/imunologia , Movimento Celular/imunologia , Fator Regulador 2 de Interferon/imunologia , Vírus Sindbis/imunologia , Infecções por Alphavirus/genética , Infecções por Alphavirus/patologia , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linfócitos B/patologia , Encefalopatias/genética , Encefalopatias/patologia , Encefalopatias/virologia , Movimento Celular/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Fator Regulador 2 de Interferon/genética , Camundongos , Camundongos Knockout
12.
J Virol ; 90(22): 10247-10258, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27581990

RESUMO

Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger antiviral protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs, repressing the translation of target mRNA, and promoting the degradation of target mRNA. We report here that murine embryonic fibroblast cells from ZAP knockout mice supported more efficient SVNI replication than wild-type cells. SVNI infection of 10-day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23-day-old weanling mice, whose immune system is more developed than that of the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor-neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wild-type mice. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance. IMPORTANCE: Sindbis virus, a prototypic member of the Alphavirus genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host antiviral factor that potently inhibits Sindbis virus replication in cell culture. We show here that infection of ZAP knockout suckling mice with an SVNI led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.


Assuntos
Infecções por Alphavirus/imunologia , Antivirais/imunologia , Vírus Sindbis/imunologia , Infecções por Alphavirus/virologia , Animais , Linhagem Celular , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Cricetinae , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/imunologia , Replicação Viral/imunologia
13.
Cell Host Microbe ; 20(2): 123-4, 2016 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-27512896

RESUMO

Polyamines play important roles in a range of cellular processes. In this issue of Cell Host & Microbe, Mounce et al. (2016) link polyamine metabolism to the interferon response and demonstrate proviral effects for polyamines. The study points to the pathway as a potential novel pan-viral therapeutic target.


Assuntos
Poliaminas
14.
J Virol ; 90(6): 3212-28, 2016 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-26739057

RESUMO

UNLABELLED: DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE: Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.


Assuntos
Proteínas Fetais/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Vírus da Febre Amarela/fisiologia , Linhagem Celular , Humanos , Proteólise
15.
Trends Cell Biol ; 25(6): 320-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25748385

RESUMO

Type I interferon (IFN) is one of the first lines of cellular defense against viral pathogens. As a result of IFN signaling, a wide array of IFN-stimulated gene (ISG) products is upregulated to target different stages of the viral life cycle. We review recent findings implicating a subset of ISGs in translational regulation of viral and host mRNAs. Translation inhibition is mediated either by binding to viral RNA or by disrupting physiological interactions or levels of the translation complex components. In addition, many of these ISGs localize to translationally silent cytoplasmic granules, such as stress granules and processing bodies, and intersect with the microRNA (miRNA)-mediated silencing pathway to regulate translation of cellular mRNAs.


Assuntos
Regulação da Expressão Gênica/genética , Interações Hospedeiro-Patógeno/imunologia , Interferons/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Transdução de Sinais/genética , Animais , Interações Hospedeiro-Patógeno/genética , Humanos
16.
J Virol ; 88(19): 11022-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25031347

RESUMO

UNLABELLED: Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. IMPORTANCE: Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artefatos , Hepacivirus/metabolismo , Hepatócitos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Hepacivirus/genética , Hepatócitos/citologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas não Estruturais Virais/genética
17.
Proc Natl Acad Sci U S A ; 110(27): 11085-90, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23776219

RESUMO

S-prenylation is an important lipid modification that targets proteins to membranes for cell signaling and vesicle trafficking in eukaryotes. As S-prenylated proteins are often key effectors for oncogenesis, congenital disorders, and microbial pathogenesis, robust proteomic methods are still needed to biochemically characterize these lipidated proteins in specific cell types and disease states. Here, we report that bioorthogonal proteomics of macrophages with an improved alkyne-isoprenoid chemical reporter enables large-scale profiling of prenylated proteins, as well as the discovery of unannotated lipidated proteins such as isoform-specific S-farnesylation of zinc-finger antiviral protein (ZAP). Notably, S-farnesylation was crucial for targeting the long-isoform of ZAP (ZAPL/PARP-13.1/zc3hav1) to endolysosomes and enhancing the antiviral activity of this immune effector. These studies demonstrate the utility of isoprenoid chemical reporters for proteomic analysis of prenylated proteins and reveal a role for protein prenylation in host defense against viral infections.


Assuntos
Prenilação de Proteína , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Lipoproteínas/química , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Dados de Sequência Molecular , Prenilação/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/genética , Ratos , Homologia de Sequência de Aminoácidos , Viroses/metabolismo , Viroses/prevenção & controle
18.
J Virol ; 86(21): 11815-32, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22915803

RESUMO

DNAJC14 is an Hsp40 family member that broadly modulates flavivirus replication. The mechanism by which DNAJC14 stoichiometrically participates in flavivirus replication complex (RC) formation is unknown; both reduced and elevated levels result in replication inhibition. Using yellow fever virus (YFV), we demonstrate that DNAJC14 redistributes and clusters with YFV nonstructural proteins via a transmembrane domain and a newly identified membrane-binding domain (MBD), which both mediate targeting to detergent-resistant membranes. Furthermore, the RC and DNAJC14 reside as part of a protein interaction network that remains after 1% Triton solubilization. Mutagenesis studies demonstrate that entry into this protein interaction network requires the DNAJC14 C-terminal self-interaction domain. Fusion of the DNAJC14 MBD and self-interaction domain with another Hsp40 family protein is sufficient to confer YFV-inhibitory activity. Our findings support a novel model of DNAJC14 action that includes specific membrane targeting of both DNAJC14 and YFV replication proteins, the formation of protein interactions, and a microdomain-specific chaperone event leading to RC formation. This process alters the properties of the RC membrane and results in the formation of a protein scaffold that maintains the RC.


Assuntos
Proteínas Fetais/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/metabolismo , Replicação Viral , Vírus da Febre Amarela/fisiologia , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/enzimologia , Proteínas Fetais/genética , Humanos , Chaperonas Moleculares/genética , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Virais/metabolismo
19.
PLoS One ; 7(5): e37398, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22615998

RESUMO

The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.


Assuntos
Alphavirus/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Interferon-alfa/farmacologia , Vírus Sindbis/efeitos dos fármacos , Animais , Linhagem Celular , Cricetinae , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Fator Regulador 2 de Interferon/farmacologia , Interferon-alfa/genética , Proteínas de Ligação a RNA , Ratos
20.
J Exp Med ; 209(4): 661-9, 2012 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-22412156

RESUMO

Effective antiviral immunity depends on the ability of infected cells or cells triggered with virus-derived nucleic acids to produce type I interferon (IFN), which activates transcription of numerous antiviral genes. However, disproportionately strong or chronic IFN expression is a common cause of inflammatory and autoimmune diseases. We describe an epigenetic mechanism that determines cell type-specific differences in IFN and IFN-stimulated gene (ISG) expression in response to exogenous signals. We identify di-methylation of histone H3 at lysine 9 (H3K9me2) as a suppressor of IFN and IFN-inducible antiviral gene expression. We show that levels of H3K9me2 at IFN and ISG correlate inversely with the scope and amplitude of IFN and ISG expression in fibroblasts and dendritic cells. Accordingly, genetic ablation or pharmacological inactivation of lysine methyltransferase G9a, which is essential for the generation of H3K9me2, resulted in phenotypic conversion of fibroblasts into highly potent IFN-producing cells and rendered these cells resistant to pathogenic RNA viruses. In summary, our studies implicate H3K9me2 and enzymes controlling its abundance as key regulators of innate antiviral immunity.


Assuntos
Epigênese Genética , Histonas/metabolismo , Interferons/biossíntese , Viroses/imunologia , Animais , Histona-Lisina N-Metiltransferase/fisiologia , Imunidade Inata , Metilação , Camundongos , Camundongos Endogâmicos C57BL
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