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1.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
2.
Cells ; 9(7)2020 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-32635598

RESUMO

The SARS-CoV-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (COVID-19). Previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. The precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. In turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are FDA-approved for the treatment of other diseases), is usually detrimental to viral replication, including SARS-CoV-2. Recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. Still, some potential therapeutic targets within this pathway, such as RAB9 and its interacting proteins, look promising considering current knowledge. Thus, the combinatory treatment of COVID-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection.


Assuntos
Autofagia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Apoptose , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/metabolismo , Betacoronavirus/classificação , Betacoronavirus/fisiologia , Proteínas do Capsídeo/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Replicação Viral/efeitos dos fármacos
3.
Nat Commun ; 11(1): 3505, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665593

RESUMO

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.


Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
4.
Nat Commun ; 11(1): 3748, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719311

RESUMO

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a ß-hairpin loop, and interconnected along the tail by the splayed ß-hairpins. By contrast, we show that the ß-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.


Assuntos
Bacteriófagos/fisiologia , Flagelos/fisiologia , Motivos de Aminoácidos , Bacteriófagos/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , DNA/genética , DNA Viral/genética , Flagelos/ultraestrutura , Herpesviridae/ultraestrutura , Multimerização Proteica , Estrutura Secundária de Proteína , Vírion/ultraestrutura , Vitrificação
5.
Proc Natl Acad Sci U S A ; 117(31): 18711-18718, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690697

RESUMO

KREMEN1 (KRM1) has been identified as a functional receptor for Coxsackievirus A10 (CV-A10), a causative agent of hand-foot-and-mouth disease (HFMD), which poses a great threat to infants globally. However, the underlying mechanisms for the viral entry process are not well understood. Here we determined the atomic structures of different forms of CV-A10 viral particles and its complex with KRM1 in both neutral and acidic conditions. These structures reveal that KRM1 selectively binds to the mature viral particle above the canyon of the viral protein 1 (VP1) subunit and contacts across two adjacent asymmetry units. The key residues for receptor binding are conserved among most KRM1-dependent enteroviruses, suggesting a uniform mechanism for receptor binding. Moreover, the binding of KRM1 induces the release of pocket factor, a process accelerated under acidic conditions. Further biochemical studies confirmed that receptor binding at acidic pH enabled CV-A10 virion uncoating in vitro. Taken together, these findings provide high-resolution snapshots of CV-A10 entry and identify KRM1 as a two-in-one receptor for enterovirus infection.


Assuntos
Proteínas do Capsídeo , Enterovirus Humano A , Proteínas de Membrana , Internalização do Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/química , Enterovirus Humano A/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Vírion/química , Vírion/metabolismo , Desenvelopamento do Vírus
6.
PLoS Pathog ; 16(6): e1008588, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584886

RESUMO

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , DNA Viral/genética , Humanos , Vírion/genética
8.
Mol Immunol ; 123: 26-31, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32388275

RESUMO

Nanoparticles are highly immunogenic due to the multivalent, repetitive antigen expression and direct activation of antigen presenting cells (APCs), key players of adaptive immune responses. Different virus-like particles (VLPs) have been used as display platforms to amplify immune responses to biologically relevant, but poorly immunogenic foreign antigens. A candidate platform based on rotavirus (RV) inner-capsid protein VP6 oligomers, such as nanotubes (T-VP6) and nanospheres (S-VP6), is also considered. Different VP6 nanostructures were compared for internalization and antigen presentation by the APCs. We found, that a lack of a high-order structures, T-VP6 and S-VP6, did not negatively affect VP6 uptake and presentation by murine bone-marrow derived dendritic cells (BMDCs) in vitro. Furthermore, T-VP6 was preferable to norovirus (NoV) VLPs for BMDC internalization resulting in significantly higher VP6-specific immune responses when T-VP6 and NoV VLP pulsed BMDCs were transferred to syngeneic mice. These results support the use of different VP6 nanostructures as foreign antigen delivery platforms.


Assuntos
Apresentação do Antígeno , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Rotavirus/fisiologia , Animais , Formação de Anticorpos , Antígenos Virais/química , Proteínas do Capsídeo/química , Células Cultivadas , Células Dendríticas/virologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Rotavirus/metabolismo , Internalização do Vírus
9.
Proc Natl Acad Sci U S A ; 117(20): 10673-10680, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358197

RESUMO

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qß for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes.


Assuntos
Bacteriófagos/genética , Proteínas do Capsídeo/metabolismo , Genoma Viral , RNA Viral/metabolismo , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Bromovirus/genética , Bromovirus/metabolismo , Bromovirus/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , RNA Viral/genética
10.
Virology ; 546: 127-132, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452411

RESUMO

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Regulação Viral da Expressão Gênica , Motivos de Aminoácidos , Proteínas do Capsídeo/genética , Dependovirus/química , Dependovirus/fisiologia , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Montagem de Vírus , Replicação Viral
11.
Int J Food Microbiol ; 323: 108592, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32315871

RESUMO

Microbial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm-1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation.


Assuntos
Bacillus subtilis/metabolismo , Bacillus subtilis/efeitos da radiação , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/efeitos da radiação , Descontaminação/métodos , Luz , Bacillus subtilis/genética , Parede Celular/metabolismo , Parede Celular/efeitos da radiação , Descontaminação/normas , Proteômica , Esporos Bacterianos/fisiologia , Esporos Bacterianos/efeitos da radiação
12.
PLoS Comput Biol ; 16(4): e1007782, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32310951

RESUMO

Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 Å resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism.


Assuntos
Proteínas do Capsídeo , Capsídeo , Vírus da Hepatite B/química , Subunidades Proteicas , Sequência de Aminoácidos , Sítios de Ligação , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Biologia Computacional/métodos , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Termodinâmica
13.
PLoS Pathog ; 16(4): e1008507, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282853

RESUMO

The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5α and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the well-characterized HIV restriction factor TRIM5α. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation, and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 can also restrict some SIV capsids. TRIM34 restriction requires TRIM5α as knockout or knockdown of TRIM5α results in a loss of antiviral activity. Through immunofluorescence studies, we show that TRIM34 and TRIM5α colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT HIV-1 capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing to restriction of HIV-1 infection in human cells.


Assuntos
Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Infecções por HIV/virologia , Soropositividade para HIV , HIV-1/metabolismo , Células HeLa , Humanos , Transcrição Reversa , Integração Viral/fisiologia
14.
Proc Natl Acad Sci U S A ; 117(19): 10286-10293, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341150

RESUMO

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA-SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA-SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N-H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA-SP1 cleavage.


Assuntos
Proteínas do Capsídeo/química , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/química , Succinatos/farmacologia , Triterpenos/farmacologia , Vírion/efeitos dos fármacos , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Vírion/genética , Vírion/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
15.
Nat Methods ; 17(5): 541-550, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313222

RESUMO

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood-brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.


Assuntos
Encéfalo/virologia , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/genética , Integrases/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Proteínas do Capsídeo/genética , Feminino , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Tropismo Viral
16.
Proc Natl Acad Sci U S A ; 117(12): 6571-6579, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156728

RESUMO

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/metabolismo , Aptâmeros de Nucleotídeos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteína 9 Associada à CRISPR/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Guia , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transcrição Genética
17.
PLoS Pathog ; 16(3): e1008428, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32187235

RESUMO

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.


Assuntos
Adaptação Fisiológica , Proteínas do Capsídeo , Enterovirus Humano A , Genoma Viral , Mutação de Sentido Incorreto , Receptores Virais , Substituição de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/metabolismo , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/metabolismo , Doença de Mão, Pé e Boca/patologia , Humanos , Receptores Virais/metabolismo , Células Vero
18.
PLoS Pathog ; 16(3): e1008386, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32208455

RESUMO

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.


Assuntos
Proteínas do Capsídeo/química , Antígenos do Grupo Sanguíneo de Lewis/química , Simulação de Acoplamento Molecular , Rotavirus/química , Proteínas do Capsídeo/metabolismo , Células HT29 , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rotavirus/metabolismo
19.
PLoS Pathog ; 16(3): e1008282, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150585

RESUMO

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/enzimologia , Proteína Quinase C-delta/metabolismo , RNA Viral/metabolismo , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Feminino , Cavalos , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos C3H , Fosforilação , Ligação Proteica , Proteína Quinase C-delta/genética , RNA Viral/genética
20.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32213611

RESUMO

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors.IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.


Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , DNA Viral , Bocavirus Humano/fisiologia , Infecções por Parvoviridae , Montagem de Vírus , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/patologia , Transdução Genética
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