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1.
J Med Microbiol ; 70(9)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34499027

RESUMO

Introduction. Zika virus (ZIKV) emerged as a public health concern on the American continent during late 2015. As the number of infected grew so did the concerns about its capability to cause long-term damage especially with the appearance of the congenital Zika syndrome (CZS). Proteins from the TAM family of receptor tyrosine kinases (RTKs) were proposed as the cellular receptors, however, due to the ability of the virus to infect a variety of cell lines different strategies to elucidate the tropism of the virus should be investigated.Hypothesis. Pseudotyping is a powerful tool to interrogate the ability of the glycoprotein (GP) to permit entry of viruses.Aim. We aimed to establish a highly tractable pseudotype model using lenti- and retro-viral backbones to investigate the entry pathway of ZIKV.Methodology. We used different glycoprotein constructs and different lenti- or retro-viral backbones, in a matrix of ratios to investigate production of proteins and functional pseudotypes.Results. Varying the ratio of backbone and glycoprotein plasmids did not yield infectious pseudotypes. Moreover, the supplementation of the ZIKV protease or the substitution of the backbone had no positive impact on the infectivity. We showed production of the proteins in producer cells implying the lack of infectious pseudotypes is due to a lack of successful glycoprotein incorporation, rather than lack of protein production.Conclusion. In line with other reports, we were unable to successfully produce infectious pseudotypes using the variety of methods described. Other strategies may be more suitable in the development of an efficient pseudotype model for ZIKV and other flaviviruses.


Assuntos
Glicoproteínas/genética , Proteínas Virais/genética , Virologia/métodos , Infecção por Zika virus/virologia , Zika virus/isolamento & purificação , Glicoproteínas/metabolismo , Humanos , Proteínas Virais/metabolismo , Internalização do Vírus , Zika virus/classificação , Zika virus/genética , Zika virus/fisiologia
2.
J Microbiol ; 59(9): 840-847, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34383247

RESUMO

Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for LysSAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.


Assuntos
Endopeptidases/metabolismo , Fagos de Staphylococcus/enzimologia , Staphylococcus aureus/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Parede Celular/metabolismo , Parede Celular/virologia , Endopeptidases/química , Endopeptidases/genética , Peptidoglicano/metabolismo , Alinhamento de Sequência , Fagos de Staphylococcus/química , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
3.
Nat Commun ; 12(1): 4710, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354070

RESUMO

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.


Assuntos
DNA Bacteriano/genética , Genes Virais , Siphoviridae/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/metabolismo , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/genética , Adenilossuccinato Sintase/metabolismo , Bacteriófagos , Pareamento de Bases , Cristalografia por Raios X , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Viral , Redes e Vias Metabólicas , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Podoviridae/classificação , Podoviridae/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Siphoviridae/classificação , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
DNA Res ; 28(4)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34390569

RESUMO

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.


Assuntos
Bacteriófagos/genética , Genoma Viral , Klebsiella pneumoniae/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Genômica , Filogenia , Análise de Sequência de DNA , Proteínas Virais/genética
5.
Nucleic Acids Res ; 49(15): 8811-8821, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365500

RESUMO

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a 'priming element' (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665-680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar 'induced-fit' mechanisms may be commonly taken by PE-containing de novo viral RdRPs.


Assuntos
Vírus da Febre Suína Clássica/enzimologia , RNA Polimerase Dependente de RNA/química , Iniciação da Transcrição Genética , Proteínas Virais/química , Cristalografia por Raios X , Flaviviridae/enzimologia , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Nucleic Acids Res ; 49(15): 8796-8810, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379778

RESUMO

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Assuntos
Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Mutação , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Proteínas Virais/genética
7.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34356005

RESUMO

The family Arteriviridae comprises enveloped RNA viruses with a linear, positive-sense genome of approximately 12.7 to 15.7 kb. The spherical, pleomorphic virions have a median diameter of 50-74 nm and include eight to eleven viral proteins. Arteriviruses infect non-human mammals in a vector-independent manner. Infections are often persistent and can either be asymptomatic or produce overt disease. Some arteriviruses are important veterinary pathogens while others infect particular species of wild rodents or African non-human primates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arteriviridae, which is available at ictv.global/report/arteriviridae.


Assuntos
Arteriviridae/classificação , Arteriviridae/genética , Filogenia , Animais , Arteriviridae/ultraestrutura , Arterivirus/classificação , Arterivirus/genética , Endocitose , Genoma Viral , Primatas , Infecções por Vírus de RNA , Proteínas Virais/genética , Vírion/classificação , Vírion/genética , Vírion/ultraestrutura , Ligação Viral , Replicação Viral
8.
PLoS Pathog ; 17(8): e1009800, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34437657

RESUMO

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.


Assuntos
Interferon beta/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/farmacologia , SARS-CoV-2/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Células Vero , Proteínas Virais/genética
9.
Nat Commun ; 12(1): 5120, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433827

RESUMO

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infected >200 million people resulting in >4 million deaths. However, temporal landscape of the SARS-CoV-2 translatome and its impact on the human genome remain unexplored. Here, we report a high-resolution atlas of the translatome and transcriptome of SARS-CoV-2 for various time points after infecting human cells. Intriguingly, substantial amount of SARS-CoV-2 translation initiates at a novel translation initiation site (TIS) located in the leader sequence, termed TIS-L. Since TIS-L is included in all the genomic and subgenomic RNAs, the SARS-CoV-2 translatome may be regulated by a sophisticated interplay between TIS-L and downstream TISs. TIS-L functions as a strong translation enhancer for ORF S, and as translation suppressors for most of the other ORFs. Our global temporal atlas provides compelling insight into unique regulation of the SARS-CoV-2 translatome and helps comprehensively evaluate its impact on the human genome.


Assuntos
COVID-19/virologia , Biossíntese de Proteínas , SARS-CoV-2/genética , Transcriptoma , Regulação Viral da Expressão Gênica , Genoma Humano , Humanos , Fases de Leitura Aberta , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
Emerg Microbes Infect ; 10(1): 1683-1690, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34348599

RESUMO

At the end of 2019, A new type of beta-CoV, SARS-CoV-2 emerged and triggered the COVID-19 pandemic, which spread overwhelmingly around the world in less than a year. However, the origin and direct ancestral viruses of SARS-CoV-2 remain unknown. RaTG13, a novel coronavirus found in bats in China's Yunnan Province, is the closest relative virus of the SARS-CoV-2 identified so far. In this study, a new SARS-CoV-2 related virus, provisionally named PrC31, was discovered in Yunnan province by retrospectively analyse bat next generation sequencing (NGS) data of intestinal samples collected in 2018. PrC31 shared 90.7% and 92.0% nucleotide identities to the genomes of SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment of PrC31 showed that several genomic regions, especially orf1a and orf8 had the highest homology with those corresponding genomic regions of SARS-CoV-2 than any other related viruses. Phylogenetic analysis indicated that PrC31 shared a common ancestor with SARS-CoV-2 in evolutionary history. The differences between the PrC31 and SARS-CoV-2 genomes were mainly manifested in the spike genes. The amino acid homology between the receptor binding domains of PrC31 and SARS-CoV-2 was only 64.2%. Importantly, recombination analysis revealed that PrC31 underwent multiple complex recombination events (including three recombination breakpoints) involving the SARS-CoV and SARS-CoV-2 sub-lineages, indicating that PrC31 evolved from yet-to-be-identified intermediate recombination strains. Combined with previous studies, it is revealed that the beta-CoVs may possess a more complex recombination mechanism than we thought.


Assuntos
Quirópteros/virologia , Recombinação Genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sequência de Aminoácidos , Animais , China , Genoma Viral , Filogenia , SARS-CoV-2/classificação , Alinhamento de Sequência , Proteínas Virais/genética
11.
Arch Virol ; 166(10): 2751-2762, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34341873

RESUMO

Ticks are important vector arthropods that transmit various pathogens to humans and other animals. Tick-borne viruses are of particular concern to public health as these are major agents of emerging and re-emerging infectious diseases. The Phenuiviridae family of tick-borne viruses is one of the most diverse groups and includes important human pathogenic viruses such as severe fever with thrombocytopenia syndrome virus. Phenuivirus-like sequences were detected during the surveillance of tick-borne viruses using RNA virome analysis from a pooled sample of Haemaphysalis formosensis ticks collected in Ehime, Japan. RT-PCR amplification and Sanger sequencing revealed the nearly complete viral genome sequence of all three segments. Comparisons of the viral amino acid sequences among phenuiviruses indicated that the detected virus shared 46%-70% sequence identity with known members of the Kaisodi group in the genus Uukuvirus. Furthermore, phylogenetic analysis of the viral proteins showed that the virus formed a cluster with the Kaisodi group viruses, suggesting that this was a novel virus, which was designated "Toyo virus" (TOYOV). Further investigation of TOYOV is needed, and it will contribute to understanding the natural history and the etiological importance of the Kaisodi group viruses.


Assuntos
Vírus de RNA de Sentido Negativo/classificação , Carrapatos/virologia , Sequência de Aminoácidos , Animais , Genoma Viral/genética , Humanos , Japão , Vírus de RNA de Sentido Negativo/genética , Vírus de RNA de Sentido Negativo/isolamento & purificação , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Proteínas Virais/genética , Viroma/genética
12.
Arch Virol ; 166(10): 2887-2894, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34347170

RESUMO

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.


Assuntos
Pseudomonas/virologia , Animais , Bacteriólise , Composição de Bases , Sequência de Bases , DNA Viral/genética , Genoma Viral/genética , Especificidade de Hospedeiro , Leite/microbiologia , Filogenia , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/fisiologia , Fagos de Pseudomonas/ultraestrutura , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/fisiologia , Siphoviridae/ultraestrutura , Proteínas Virais/genética , Vírion/ultraestrutura
13.
Arch Virol ; 166(10): 2901-2904, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363533

RESUMO

The complete genomic sequence of scorzonera virus A (SCoVA) from a Scorzonera austriaca Willd. plant in South Korea was determined by high-throughput sequencing and confirmed by Sanger sequencing. The SCoVA genome contains 9867 nucleotides, excluding the 3'-terminal poly(A) tail. The SCoVA genome structure is typical of potyviruses and contains a single open reading frame encoding a large putative polyprotein of 3168 amino acids. Pairwise comparison analysis of the complete genome and polyprotein sequences of SCoVA with those of other potyviruses showed that they shared the highest nucleotide and amino acid sequences identity (54.47% and 49.57%, respectively) with those of lettuce mosaic virus (GenBank accession number KJ161185). Phylogenetic analysis of the amino acid sequence of the polyprotein confirmed that SCoVA belongs to the genus Potyvirus. These findings suggest that SCoVA should be considered a novel member of the genus Potyvirus, family Potyviridae.


Assuntos
Genoma Viral/genética , Potyvirus/genética , Scorzonera/virologia , Sequência de Aminoácidos , Sequência de Bases , Fases de Leitura Aberta/genética , Filogenia , Poliproteínas/genética , Potyvirus/classificação , Potyvirus/isolamento & purificação , RNA Viral/genética , República da Coreia , Proteínas Virais/genética
14.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372577

RESUMO

A novel Enterobacter cloacae phage, EC151, was isolated and characterized. Electron microscopy revealed that EC151 has a siphovirus-like virion morphology. The EC151 nucleotide sequence shows limited similarity to other phage genomes deposited in the NCBI GenBank database. The size of the EC151 genome is 60,753 bp and contains 58 putative genes. Thirty-nine of them encode proteins of predicted function, 18 are defined as hypothetical proteins, and one ORF identifies as the tRNA-Ser-GCT-encoding gene. Six ORFs were predicted to be members of the deazaguanine DNA modification pathway, including the preQ0 transporter. Comparative proteomic phylogenetic analysis revealed that phage EC151 represents a distinct branch within a group of sequences containing clades formed by members of the Seuratvirus, Nonagvirus, and Vidquintavirus genera. In addition, the EC151 genome showed gene synteny typical of the Seuratvirus, Nonagvirus, and Nipunavirus phages. The average genetic distances of EC151/Seuratvirus, EC151/Nonagvirus, and EC151/Vidquintavirus are approximately equal to those between the Seuratvirus, Nonagvirus, and Vidquintavirus genera (~0.7 substitutions per site). Therefore, EC151 may represent a novel genus within the Siphoviridae family. The origin of the deazaguanine DNA modification pathway in the EC151 genome can be traced to Escherichia phages from the Seuratvirus genus.


Assuntos
Bacteriófagos/genética , Enterobacter cloacae/genética , Enterobacter cloacae/virologia , DNA Viral/genética , Enterobacter cloacae/metabolismo , Genoma Viral/genética , Genômica , Especificidade de Hospedeiro , Filogenia , Proteômica , Siphoviridae/genética , Proteínas Virais/genética , Vírion/metabolismo
15.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372584

RESUMO

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.


Assuntos
Bacteriólise/fisiologia , Micobacteriófagos/metabolismo , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases , Hidrólise , Mycobacterium/metabolismo , Mycobacterium/virologia , Peptidoglicano/metabolismo , Ligação Proteica
16.
Genes (Basel) ; 12(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34356077

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19), by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly developed into a worldwide pandemic. Mutations in the SARS-CoV-2 genome may affect various aspects of the disease including fatality ratio. In this study, 553,518 SARS-CoV-2 genome sequences isolated from patients from continents for the period 1 December 2020 to 15 March 2021 were comprehensively analyzed and a total of 82 mutations were identified concerning the reference sequence. In addition, associations between the mutations and the case fatality ratio (CFR), cases per million and deaths per million, were examined. The mutations having the highest frequencies among different continents were Spike_D614G and NSP12_P323L. Among the identified mutations, NSP2_T153M, NSP14_I42V and Spike_L18F mutations showed a positive correlation to CFR. While the NSP13_Y541C, NSP3_T73I and NSP3_Q180H mutations demonstrated a negative correlation to CFR. The Spike_D614G and NSP12_P323L mutations showed a positive correlation to deaths per million. The NSP3_T1198K, NS8_L84S and NSP12_A97V mutations showed a significant negative correlation to deaths per million. The NSP12_P323L and Spike_D614G mutations showed a positive correlation to the number of cases per million. In contrast, NS8_L84S and NSP12_A97V mutations showed a negative correlation to the number of cases per million. In addition, among the identified clades, none showed a significant correlation to CFR. The G, GR, GV, S clades showed a significant positive correlation to deaths per million. The GR and S clades showed a positive correlation to number of cases per million. The clades having the highest frequencies among continents were G, followed by GH and GR. These findings should be taken into consideration during epidemiological surveys of the virus and vaccine development.


Assuntos
Teste para COVID-19 , COVID-19/genética , COVID-19/mortalidade , Mutação , SARS-CoV-2/genética , Proteínas Virais/genética , Feminino , Humanos , Masculino , SARS-CoV-2/patogenicidade
17.
PLoS One ; 16(8): e0256141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407143

RESUMO

SARS-CoV-2 requires serine protease, transmembrane serine protease 2 (TMPRSS2), and cysteine proteases, cathepsins B, L (CTSB/L) for entry into host cells. These host proteases activate the spike protein and enable SARS-CoV-2 entry. We herein performed genomic-guided gene set enrichment analysis (GSEA) to identify upstream regulatory elements altering the expression of TMPRSS2 and CTSB/L. Further, medicinal compounds were identified based on their effects on gene expression signatures of the modulators of TMPRSS2 and CTSB/L genes. Using this strategy, estradiol and retinoic acid have been identified as putative SARS-CoV-2 alleviation agents. Next, we analyzed drug-gene and gene-gene interaction networks using 809 human targets of SARS-CoV-2 proteins. The network results indicate that estradiol interacts with 370 (45%) and retinoic acid interacts with 251 (31%) human proteins. Interestingly, a combination of estradiol and retinoic acid interacts with 461 (56%) of human proteins, indicating the therapeutic benefits of drug combination therapy. Finally, molecular docking analysis suggests that both the drugs bind to TMPRSS2 and CTSL with the nanomolar to low micromolar affinity. The results suggest that these drugs can simultaneously target both the entry pathways of SARS-CoV-2 and thus can be considered as a potential treatment option for COVID-19.


Assuntos
Catepsina B/genética , Catepsina L/genética , Estradiol/farmacologia , Genômica/métodos , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Tretinoína/farmacologia , Catepsina B/química , Catepsina L/química , Bases de Dados Genéticas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos
18.
Diagn Microbiol Infect Dis ; 101(2): 115458, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34274751

RESUMO

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Teste de Ácido Nucleico para COVID-19/normas , Fluorescência , Temperatura Alta , Humanos , Reação em Cadeia da Polimerase Multiplex , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes , Proteínas Virais/genética
19.
Genes (Basel) ; 12(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202172

RESUMO

A signal analysis of the complete genome sequenced for coronavirus variants of concern-B.1.1.7 (Alpha), B.1.135 (Beta) and P1 (Gamma)-and coronavirus variants of interest-B.1.429-B.1.427 (Epsilon) and B.1.525 (Eta)-is presented using open GISAID data. We deal with a certain new type of finite alternating sum series having independently distributed terms associated with binary (0,1) indicators for the nucleotide bases. Our method provides additional information to conventional similarity comparisons via alignment methods and Fourier Power Spectrum approaches. It leads to uncover distinctive patterns regarding the intrinsic data organization of complete genomics sequences according to its progression along the nucleotide bases position. The present new method could be useful for the bioinformatics surveillance and dynamics of coronavirus genome variants.


Assuntos
Biologia Computacional/métodos , Genoma Viral , SARS-CoV-2/genética , Proteínas Virais/genética , Coronavirus/genética , Humanos , SARS-CoV-2/isolamento & purificação
20.
Arch Virol ; 166(9): 2369-2386, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34216267

RESUMO

Rotaviruses are segmented double-stranded RNA viruses with a high frequency of gene reassortment, and they are a leading cause of global diarrheal deaths in children less than 5 years old. Two-thirds of rotavirus-associated deaths occur in low-income countries. Currently, the available vaccines in developing countries have lower efficacy in children than those in developed countries. Due to added safety concerns and the high cost of current vaccines, there is a need to develop cost-effective next-generation vaccines with improved safety and efficacy. The reverse genetics system (RGS) is a powerful tool for investigating viral protein functions and developing novel vaccines. Recently, an entirely plasmid-based RGS has been developed for several rotaviruses, and this technological advancement has significantly facilitated novel rotavirus research. Here, we review the recently developed RGS platform and discuss its application in studying infection biology, gene reassortment, and development of vaccines against rotavirus disease.


Assuntos
Genética Reversa , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/genética , Rotavirus/genética , Animais , Pré-Escolar , Análise Custo-Benefício , Diarreia/prevenção & controle , Diarreia/virologia , Interações Hospedeiro-Patógeno , Humanos , Plasmídeos , RNA Viral/genética , Vacinas contra Rotavirus/economia , Proteínas Virais/genética
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