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1.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32994342

RESUMO

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de Vírus
2.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32788194

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Vírus da Leucemia Murina , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Internalização do Vírus
3.
Virology ; 548: 124-131, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838933

RESUMO

SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal.


Assuntos
Cromatina/virologia , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Vírus 40 dos Símios/metabolismo , Fator de Transcrição Sp1/metabolismo , Vírion/metabolismo , Cromatina/genética , Cromatina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Infecções por Polyomavirus/genética , Ligação Proteica , Vírus 40 dos Símios/genética , Fator de Transcrição Sp1/genética , Vírion/genética
4.
Arch Virol ; 165(10): 2349-2353, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32743696

RESUMO

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.


Assuntos
Arachis/virologia , Genoma Viral , Filogenia , Potyviridae/genética , RNA Viral/genética , Proteínas Virais/genética , Brasil , Fases de Leitura Aberta , Doenças das Plantas/virologia , Folhas de Planta/virologia , Potyviridae/classificação , Potyviridae/isolamento & purificação , Potyviridae/ultraestrutura , Vírion/genética , Vírion/isolamento & purificação , Vírion/ultraestrutura
5.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641479

RESUMO

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.


Assuntos
Citidina Desaminase/genética , Regulação Viral da Expressão Gênica , Produtos do Gene gag/genética , Leucemia Experimental/genética , Vírus da Leucemia Murina de Moloney/genética , Infecções por Retroviridae/genética , Infecções Tumorais por Vírus/genética , Processamento Alternativo , Animais , Capsídeo/metabolismo , Citidina Desaminase/deficiência , Produtos do Gene gag/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/virologia , Camundongos , Camundongos Knockout , Vírus da Leucemia Murina de Moloney/metabolismo , Vírus da Leucemia Murina de Moloney/patogenicidade , Células NIH 3T3 , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Transdução de Sinais , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologia , Vírion/genética , Vírion/metabolismo , Vírion/patogenicidade , Replicação Viral
6.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611759

RESUMO

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.


Assuntos
Ebolavirus/efeitos dos fármacos , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas Virais de Fusão/antagonistas & inibidores , Vírion/efeitos dos fármacos , Animais , Sítios de Ligação , Bioensaio , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacologia , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacologia , Toremifeno/química , Toremifeno/farmacologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
7.
BMC Infect Dis ; 20(1): 488, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32646445

RESUMO

BACKGROUND: Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system. METHODS: To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively. RESULTS: The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I. CONCLUSIONS: Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases.


Assuntos
Células Epiteliais/virologia , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Mucosa Respiratória/patologia , Infecções Respiratórias/diagnóstico , Adolescente , Proteínas do Capsídeo/genética , Polaridade Celular , Células Cultivadas , Criança , Pré-Escolar , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Vírion/genética , Replicação Viral , Sequenciamento Completo do Genoma
8.
Infect Genet Evol ; 84: 104451, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640381

RESUMO

WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Proteínas do Nucleocapsídeo/química , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Vírion/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Doxiciclina/química , Doxiciclina/farmacologia , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinoxalinas/química , Quinoxalinas/farmacologia , Rutina/química , Rutina/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Simeprevir/química , Simeprevir/farmacologia , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Vírion/genética , Vírion/metabolismo
9.
J Pak Med Assoc ; 70(Suppl 3)(5): S38-S43, 2020 May.
Artigo em Inglês | MEDLINE | ID: covidwho-592548

RESUMO

COVID-19 has taken the world by storm in the ongoing pandemic. The virus responsible for COVID-19 disease is 'severe acute respiratory syndrome coronavirus-2' SARS-CoV-2, an enveloped RNA beta-coronavirus from the family Coronaviridae. There have been similar beta-coronavirus disease outbreaks previously: Severe acute respiratory syndrome (SARS - 2002) and Middle East respiratory syndrome (MERS - 2012) epidemics. SARS-CoV-2 origins have been traced to bat reservoirs. A virus with a high capacity for mutation, SARS-CoV-2 poses unique challenges both in the current form of disease control and management, while also leaving the door open for future novel diseases and pandemics. An understanding of the virion structure and genomic organisation will help us in understanding their origins and likely course of future evolution. Moreover, novel cost-effective methodologies for genetic surveillance may help in mitigating the emergence of these viral infections in future. In this manuscript, the authors have detailed the unique aspects of the SARS-CoV-2 virus genome and its clinical implications.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Animais , Ásia , Quirópteros/virologia , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Taxa de Mutação , Pandemias , Pneumonia Viral/transmissão , RNA Viral/genética , Vírion/genética
10.
J Pak Med Assoc ; 70(Suppl 3)(5): S38-S43, 2020 May.
Artigo em Inglês | MEDLINE | ID: covidwho-609354

RESUMO

COVID-19 has taken the world by storm in the ongoing pandemic. The virus responsible for COVID-19 disease is 'severe acute respiratory syndrome coronavirus-2' SARS-CoV-2, an enveloped RNA beta-coronavirus from the family Coronaviridae. There have been similar beta-coronavirus disease outbreaks previously: Severe acute respiratory syndrome (SARS - 2002) and Middle East respiratory syndrome (MERS - 2012) epidemics. SARS-CoV-2 origins have been traced to bat reservoirs. A virus with a high capacity for mutation, SARS-CoV-2 poses unique challenges both in the current form of disease control and management, while also leaving the door open for future novel diseases and pandemics. An understanding of the virion structure and genomic organisation will help us in understanding their origins and likely course of future evolution. Moreover, novel cost-effective methodologies for genetic surveillance may help in mitigating the emergence of these viral infections in future. In this manuscript, the authors have detailed the unique aspects of the SARS-CoV-2 virus genome and its clinical implications.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Animais , Ásia , Quirópteros/virologia , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Taxa de Mutação , Pandemias , Pneumonia Viral/transmissão , RNA Viral/genética , Vírion/genética
11.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32522855

RESUMO

The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function.IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Empacotamento do DNA/fisiologia , Peptídeos/metabolismo , Vírion/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , DNA Viral , Vetores Genéticos , Genoma Viral , Humanos , Ligantes , Vírion/genética
12.
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
13.
Arch Virol ; 165(11): 2723-2731, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32583077

RESUMO

Established in 2016, the family Pleolipoviridae comprises globally distributed archaeal viruses that produce pleomorphic particles. Pseudo-spherical enveloped virions of pleolipoviruses are membrane vesicles carrying a nucleic acid cargo. The cargo can be either a single-stranded or double-stranded DNA molecule, making this group the first family introduced in the 10th Report on Virus Taxonomy including both single-stranded and double-stranded DNA viruses. The length of the genomes is approximately 7-17 kilobase pairs, or kilonucleotides in the case of single-stranded molecules. The genomes are circular single-stranded DNA, circular double-stranded DNA, or linear double-stranded DNA molecules. Currently, eight virus species and seven proposed species are classified in three genera: Alphapleolipovirus (five species), Betapleolipovirus (nine species), and Gammapleolipovirus (one species). Here, we summarize the updated taxonomy of the family Pleolipoviridae to reflect recent advances in this field, with the focus on seven newly proposed species in the genus Betapleolipovirus: Betapleolipovirus HHPV3, HHPV4, HRPV9, HRPV10, HRPV11, HRPV12, and SNJ2.


Assuntos
Archaea/virologia , Vírus de Archaea/classificação , Vírus de Archaea/isolamento & purificação , DNA Viral/genética , Genoma Viral , Vírion/genética
14.
J Pak Med Assoc ; 70(Suppl 3)(5): S38-S43, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32515376

RESUMO

COVID-19 has taken the world by storm in the ongoing pandemic. The virus responsible for COVID-19 disease is 'severe acute respiratory syndrome coronavirus-2' SARS-CoV-2, an enveloped RNA beta-coronavirus from the family Coronaviridae. There have been similar beta-coronavirus disease outbreaks previously: Severe acute respiratory syndrome (SARS - 2002) and Middle East respiratory syndrome (MERS - 2012) epidemics. SARS-CoV-2 origins have been traced to bat reservoirs. A virus with a high capacity for mutation, SARS-CoV-2 poses unique challenges both in the current form of disease control and management, while also leaving the door open for future novel diseases and pandemics. An understanding of the virion structure and genomic organisation will help us in understanding their origins and likely course of future evolution. Moreover, novel cost-effective methodologies for genetic surveillance may help in mitigating the emergence of these viral infections in future. In this manuscript, the authors have detailed the unique aspects of the SARS-CoV-2 virus genome and its clinical implications.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Animais , Ásia , Quirópteros/virologia , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Taxa de Mutação , Pandemias , Pneumonia Viral/transmissão , RNA Viral/genética , Vírion/genética
15.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376624

RESUMO

Little is known about the human cytomegalovirus (HCMV) tegument protein UL88. Large-scale genomic studies have reported disparate results for UL88-null viruses, reporting both no phenotype and a >1-log decrease in virus titers. UL88 has also been reported to interact with UL69 and UL48, but the functional relevance of this interaction is unknown. Here, we report that UL88, which is conserved among different viral strains, is dispensable for production of infectious HCMV virions in multiple HCMV strains and cell types. However, the specific infectivity of HCMV virions suffers in the absence of UL88, as more genomes are required per PFU. This may be a result of altered virion tegument protein composition, as Western blot analysis shows a significant reduction in the tegument levels of pp71, UL47, and UL48 in viruses lacking UL88. While an interaction between UL88 and UL48 has previously been reported, we show that UL88 can interact with UL47; however, UL88 does not appear to be part of a stable complex consisting of UL47 and UL48. These findings identify an important role for UL88 in incorporating the viral proteins UL47 and UL48 into the virion tegument layer.IMPORTANCE A better understanding of the role and functions of tegument proteins in HCMV, many of which remain uncharacterized, will contribute to our understanding of the biology of HCMV. The virus has a large genome, greater than 230 kb, and functional annotation of these genes is important for identifying novel targets for improving therapeutic intervention. This study identifies a role for a viral tegument protein with unknown function, UL88, in maintaining the proper tegument composition of HCMV virions. Virions produced in the absence of UL88 exhibit decreased fitness and require more genomes per infectious unit.


Assuntos
Citomegalovirus/metabolismo , Genoma Viral , Proteínas Virais/metabolismo , Vírion/metabolismo , Citomegalovirus/genética , Humanos , Células THP-1 , Proteínas Virais/genética , Vírion/genética
18.
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
19.
Sci Rep ; 10(1): 7181, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32346128

RESUMO

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.


Assuntos
Infecções por Herpesviridae , Mardivirus/metabolismo , Doenças das Aves Domésticas , Proteínas Virais/metabolismo , Vírion/metabolismo , Liberação de Vírus , Animais , Células Cultivadas , Patos , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/transmissão , Infecções por Herpesviridae/veterinária , Mardivirus/genética , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/transmissão , Proteínas Virais/genética , Vírion/genética
20.
Sci Rep ; 10(1): 6563, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300201

RESUMO

When rinderpest virus (RPV) was declared eradicated in 2011, the only remaining samples of this once much-feared livestock virus were those held in various laboratories. In order to allow the destruction of our institute's stocks of RPV while maintaining the ability to recover the various viruses if ever required, we have determined the full genome sequence of all our distinct samples of RPV, including 51 wild type viruses and examples of three different types of vaccine strain. Examination of the sequences of these virus isolates has shown that the African isolates form a single disparate clade, rather than two separate clades, which is more in accord with the known history of the virus in Africa. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the M and F protein coding sequences, and shown that, for more than half the genomes sequenced, translation of the F protein requires translational frameshift or non-standard translation initiation. Curiously, the clade containing the lapinised vaccine viruses that were developed originally in Korea appears to be more similar to the known African viruses than to any other Asian viruses.


Assuntos
Vírus da Peste Bovina/genética , Vírus da Peste Bovina/isolamento & purificação , Vacinas Virais/genética , Sequenciamento Completo do Genoma , Sequência de Bases , DNA Complementar/genética , Biblioteca Gênica , Genoma Viral , Filogenia , RNA Viral/genética , Vírion/genética
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