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1.
Turk J Med Sci ; 50(SI-1): 549-556, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32293832

RESUMO

Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 (COVID-19). COVID-19's first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/epidemiologia , Coronavirus/classificação , Pneumonia Viral/epidemiologia , Animais , China/epidemiologia , Coronavirus Humano 229E , Coronavirus Humano OC43 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteínas do Nucleocapsídeo/química , Pandemias , Peptidil Dipeptidase A/química , Vírus da SARS , Glicoproteína da Espícula de Coronavírus/química , Turquia/epidemiologia , Proteínas da Matriz Viral/química , Montagem de Vírus , Internalização do Vírus
2.
Nat Commun ; 11(1): 632, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005813

RESUMO

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.


Assuntos
Desaminase APOBEC-3G/química , Infecções por HIV/enzimologia , HIV-1/fisiologia , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismo , Animais , Dimerização , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Macaca mulatta , Domínios Proteicos , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus , Replicação Viral
3.
PLoS One ; 15(2): e0228572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045432

RESUMO

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.


Assuntos
Vacinas contra Parainfluenza/genética , Vírus da Parainfluenza 3 Humana/imunologia , Vacinas contra Vírus Sincicial Respiratório/genética , Proteínas Virais de Fusão/genética , Montagem de Vírus , Administração Intranasal , Animais , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Imunogenicidade da Vacina , Macaca mulatta , Mesocricetus , Vacinas contra Parainfluenza/administração & dosagem , Vacinas contra Parainfluenza/imunologia , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/fisiologia , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Células Vero , Proteínas Virais de Fusão/metabolismo
4.
Nat Chem Biol ; 16(3): 231-239, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32080621

RESUMO

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, we will discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.


Assuntos
Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica
5.
PLoS Pathog ; 16(1): e1008271, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905231

RESUMO

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.


Assuntos
RNA Viral/fisiologia , Tombusviridae/genética , Transativadores/fisiologia , Cucumis sativus , Evolução Molecular , Genoma Viral , Conformação de Ácido Nucleico , RNA Viral/química , Relação Estrutura-Atividade , Tombusviridae/fisiologia , Montagem de Vírus
6.
PLoS One ; 15(1): e0228028, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945138

RESUMO

Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We've also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we've used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn't seem to pose much of a further barrier to DNA mixing and so it's likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses.


Assuntos
Replicação do DNA , DNA Viral/biossíntese , Recombinação Genética , Vírus Vaccinia , Vírion/fisiologia , Montagem de Vírus , Animais , Linhagem Celular , Citosol/imunologia , Retículo Endoplasmático/imunologia , Vírus Vaccinia/genética , Vírus Vaccinia/fisiologia
7.
PLoS Pathog ; 16(1): e1008277, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31986188

RESUMO

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.


Assuntos
Anemia Infecciosa Equina/metabolismo , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Anemia Infecciosa Equina/fisiologia , Ácido Fítico/metabolismo , Vírion/fisiologia , Sequência de Aminoácidos , Animais , Tomografia com Microscopia Eletrônica , Anemia Infecciosa Equina/virologia , Produtos do Gene gag/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , HIV-1/ultraestrutura , Cavalos , Interações Hospedeiro-Patógeno , Vírus da Anemia Infecciosa Equina/química , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/ultraestrutura , Alinhamento de Sequência , Vírion/genética , Vírion/ultraestrutura , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
8.
PLoS Pathog ; 16(1): e1008264, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999790

RESUMO

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Cromatina/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Provírus/fisiologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Provírus/genética , Ativação Viral , Montagem de Vírus , Latência Viral
9.
Mol Plant Microbe Interact ; 33(1): 6-17, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31454296

RESUMO

Plum pox virus, the agent that causes sharka disease, is among the most important plant viral pathogens, affecting Prunus trees across the globe. The fabric of interactions that the virus is able to establish with the plant regulates its life cycle, including RNA uncoating, translation, replication, virion assembly, and movement. In addition, plant-virus interactions are strongly conditioned by host specificities, which determine infection outcomes, including resistance. This review attempts to summarize the latest knowledge regarding Plum pox virus-host interactions, giving a comprehensive overview of their relevance for viral infection and plant survival, including the latest advances in genetic engineering of resistant species.


Assuntos
Interações Hospedeiro-Patógeno , Vírus Eruptivo da Ameixa , Prunus , Resistência à Doença/genética , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Vírus Eruptivo da Ameixa/fisiologia , Prunus/genética , Prunus/virologia , Montagem de Vírus
10.
Arch Virol ; 165(2): 407-412, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31811441

RESUMO

In this study, two aspects of the ultrastructure of white spot syndrome virus (WSSV) were identified: (i) The virus nucleocapsids were disassembled, and transmission electron microscopy (TEM) image analysis confirmed that the nucleocapsids were composed of stacked ring segments rather than the usual helix system, with each ring segment consisting of three rows of subunits linked by filaments. (ii) In addition, the morphological characteristics of virus self-assembly at different stages were observed, and two different enveloping morphologies were found, implying that the virion matures through two distinct envelopments. Thus, we propose a viral membrane assembly process for WSSV virion.


Assuntos
Nucleocapsídeo/ultraestrutura , Montagem de Vírus , Vírus da Síndrome da Mancha Branca 1/fisiologia , Vírus da Síndrome da Mancha Branca 1/ultraestrutura , Microscopia Eletrônica de Transmissão
11.
J Agric Food Chem ; 68(2): 471-484, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31841334

RESUMO

Plant diseases seriously affect crop yield and quality and are difficult to control. Marine natural products (MNPs) have become an important source of drug candidates with new biological mechanisms. Marine natural product essramycin (1) was found to have good anti-tobacco mosaic virus (TMV) and anti-phytopathogenic fungus activities for the first time. A series of essramycin derivatives were designed, synthesized, and evaluated for their bioactivity. Most of these compounds exhibited antiviral effects that are greater than that of the control ribavirin. Compounds 7e and 8f displayed antiviral activities that are greater than that of ningnanmycin (the most widely used antiviral agent at present), thus emerging as novel antiviral lead compounds. As the lead compound, 7e was selected for further antiviral mechanism research. The results indicated that 7e could inhibit virus assembly and promote 20S disk protein aggregation. Fungicidal activity tests against 14 kinds of phytopathogenic fungi revealed that essramycin analogues displayed broad-spectrum fungicidal activities. Compound 5b displayed more than 50% inhibition rate against most of the 14 kinds of phytopathogenic fungi at 50 µg/mL. The current research lays a solid foundation for the application of essramycin alkaloids in crop protection.


Assuntos
Alcaloides/química , Antivirais/química , Fungicidas Industriais/química , Pirimidinonas/química , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Triazóis/química , Alcaloides/farmacologia , Antivirais/farmacologia , Fungos/efeitos dos fármacos , Fungos/fisiologia , Fungicidas Industriais/farmacologia , Estrutura Molecular , Doenças das Plantas/microbiologia , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Vírus do Mosaico do Tabaco/fisiologia , Triazóis/farmacologia , Montagem de Vírus/efeitos dos fármacos
12.
Emerg Microbes Infect ; 8(1): 1563-1573, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31672101

RESUMO

The episomal structures of all human bocavirus (HBoV) genotypes have been deciphered, including the circular genome of HBoV2 (HBoV2-C1). To discern the role of the circular HBoV2 genome, three distinct linearized HBoV2-C1 genomes were cloned into pBlueScript SKII(+) to obtain pBlueScript HBoV2 5043-5042 (retaining all secondary structures), pBlueScript-HBoV2 5075-5074 (retaining hairpin number 2 and the 5' terminal structure), and pBlueScript-HBoV2 5220-5219 (retaining only the 5' terminal structure at the 5' -genome end). The recombinant plasmids were separately transfected HEK293 cells, revealing that more HBoV2 DNA had accumulated in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells at 72 h post-transfection, as determined by real-time PCR. However, more mRNA was transcribed by pBlueScript-HBoV2 5075-5074 than by the other constructs, as determined by dot-blot hybridization and RNAscope. No significant differences in NS1-70 protein expression were observed among the three HBoV2 genomic clones. However, electron microscopy showed that HBoV2 virus particles were only present in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells. By using three hetero-recombinant HBoV2 genomic clones in HEK293 transfected cells, only the genome with intact secondary structures produced virus particles, suggesting that retaining these structures in a circular genome is important for HBoV2 DNA replication and virus assembly.


Assuntos
DNA Circular/genética , DNA Viral/genética , Bocavirus Humano/genética , Infecções por Parvoviridae/virologia , RNA não Traduzido/genética , Recombinação Genética , Montagem de Vírus , Replicação do DNA , DNA Circular/química , DNA Circular/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Genoma Viral , Genômica , Genótipo , Células HEK293 , Bocavirus Humano/química , Bocavirus Humano/fisiologia , Humanos , Conformação de Ácido Nucleico , Filogenia , RNA não Traduzido/química , RNA não Traduzido/metabolismo
13.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
14.
Nat Commun ; 10(1): 4471, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578335

RESUMO

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly.


Assuntos
Bacteriófagos/genética , Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Genoma Viral/genética , Instabilidade Genômica/genética , Vírion/genética , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , DNA Viral/química , DNA Viral/genética , DNA Viral/ultraestrutura , Temperatura Alta , Modelos Moleculares , Thermus thermophilus/virologia , Vírion/química , Vírion/ultraestrutura , Montagem de Vírus/genética
15.
Nat Commun ; 10(1): 4840, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649265

RESUMO

Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.


Assuntos
Bacteriófagos/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Montagem de Vírus , Bacteriófagos/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas Estruturais Virais/metabolismo , Proteínas Estruturais Virais/ultraestrutura
16.
Soft Matter ; 15(36): 7166-7172, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31483421

RESUMO

The efficient construction of a protective protein shell or capsid is one of the most crucial steps in the replication cycle of a virus. The formation of the simplest capsid typically proceeds by the spontaneous assembly of identical building blocks. This process can also be achieved in vitro even in the absence of genetic material, thus opening the door to the production of artificial viral cages for a myriad of applications. In this work, we analyze the efficiency and the kinetic peculiarities of this self-assembly process using Brownian Dynamics simulations. We use a minimal model that considers identical assembly units and is able to reproduce successfully the correct final architecture of spherical capsids. The selection of a specific size and structure is achieved by changing a single parameter that imposes an angular anisotropy on the interaction. We analyze how the geometrical constraints of the interaction affect the efficiency of the assembly. We find that the optimal conditions for an efficient assembly from a kinetic point of view strongly depart from the lowest capsid energy corresponding to the minimum of the potential energy landscape. Our work illustrates the important differences between the equilibrium and dynamic characteristics of viral self-assembly, and provides important insights on how to design specific interactions for a successful assembly of artificial viral cages.


Assuntos
Capsídeo/química , Simulação por Computador , Modelos Químicos , Montagem de Vírus , Proteínas do Capsídeo/química , Cinética , Tamanho da Partícula , Propriedades de Superfície , Termodinâmica
17.
World J Gastroenterol ; 25(32): 4580-4597, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31528088

RESUMO

Chronic delta hepatitis is the most severe form of viral hepatitis affecting nearly 65 million people worldwide. Individuals with this devastating illness are at higher risk for developing cirrhosis and hepatocellular carcinoma. Delta virus is a defective RNA virus that requires hepatitis B surface antigen for propagation in humans. Infection can occur in the form of a co-infection with hepatitis B, which can be self-limiting, vs superinfection in a patient with established hepatitis B infection, which often leads to chronicity in majority of cases. Current noninvasive tools to assess for advanced liver disease have limited utility in delta hepatitis. Guidelines recommend treatment with pegylated interferon, but this is limited to patients with compensated disease and is efficacious in about 30% of those treated. Due to limited treatment options, novel agents are being investigated and include entry, assembly and export inhibitors of viral particles in addition to stimulators of the host immune response. Future clinical trials should take into consideration the interaction of hepatitis B and hepatitis D as suppression of one virus can lead to the activation of the other. Also, surrogate markers of treatment efficacy have been proposed.


Assuntos
Antivirais/uso terapêutico , Coinfecção/tratamento farmacológico , Hepatite B Crônica/tratamento farmacológico , Hepatite D Crônica/tratamento farmacológico , Superinfecção/tratamento farmacológico , Terapias em Estudo/métodos , Antivirais/farmacologia , Coinfecção/epidemiologia , Coinfecção/virologia , Quimioterapia Combinada/métodos , Carga Global da Doença , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Hepatite D Crônica/epidemiologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Literatura de Revisão como Assunto , Superinfecção/epidemiologia , Superinfecção/virologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Resultado do Tratamento , Montagem de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
18.
Nucleic Acids Res ; 47(18): 9818-9828, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31396619

RESUMO

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA-gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a 'Z'-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.


Assuntos
Fagos Bacilares/genética , Empacotamento do DNA/genética , RNA Viral/genética , Proteínas Virais/genética , Difosfato de Adenosina/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Sítios de Ligação , Cristalografia por Raios X , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Conformação de Ácido Nucleico , RNA Viral/química , Espalhamento a Baixo Ângulo , Transdução de Sinais/genética , Proteínas Virais/química , Montagem de Vírus/genética
19.
Nucleic Acids Res ; 47(17): 9231-9242, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31396624

RESUMO

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.


Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , DNA Viral/genética , Proteínas do Core Viral/genética , Genoma Viral/genética , Humanos , Proteínas Virais/genética , Vírion/genética , Montagem de Vírus
20.
PLoS Comput Biol ; 15(8): e1006602, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31433804

RESUMO

The formation of a membrane-enveloped virus starts with the assembly of a curved layer of capsid proteins lining the interior of the plasma membrane (PM) of the host cell. This layer develops into a spherical shell (capsid) enveloped by a lipid-rich membrane. In many cases, the budding process stalls prior to the release of the virus. Recently, Brownian dynamics simulations of a coarse-grained model system reproduced protracted pausing and stalling, which suggests that the origin of pausing/stalling is to be found in the physics of the budding process. Here, we propose that the pausing/stalling observed in the simulations can be understood as a purely kinetic phenomenon associated with the neck geometry. A geometrical potential energy barrier develops during the budding that must be overcome by capsid proteins diffusing along the membrane prior to incorporation into the capsid. The barrier is generated by a conflict between the positive Gauss curvature of the assembling capsid and the negative Gauss curvature of the neck region. A continuum theory description is proposed and is compared with the Brownian simulations of the budding of enveloped viruses.


Assuntos
Modelos Biológicos , Liberação de Vírus/fisiologia , Animais , Fenômenos Biofísicos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/fisiologia , Biologia Computacional , Simulação por Computador , Glicoproteínas/química , Glicoproteínas/fisiologia , HIV-1/química , HIV-1/fisiologia , HIV-1/ultraestrutura , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Cinética , Lipídeos de Membrana/química , Lipídeos de Membrana/fisiologia , Conformação Proteica , Montagem de Vírus/fisiologia
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