scholarly journals Identification of Adomavirus Virion Proteins

2018 ◽  
Author(s):  
Nicole L. Welch ◽  
Michael J. Tisza ◽  
Gabriel J. Starrett ◽  
Anna K. Belford ◽  
Diana V. Pastrana ◽  
...  
Keyword(s):  
Dna Helicase ◽  
Amino Acid Sequences ◽  
Host Cells ◽  
Structural Proteins ◽  
Replicase Gene ◽  
Virion Protein ◽  
Virus Family ◽  
Animal Virus ◽  
Tumor Viruses ◽  
Emerging Virus

AbstractAdenoviruses, papillomaviruses, and polyomaviruses are collectively known as small DNA tumor viruses. Although it has long been recognized that small DNA tumor virus oncoproteins and capsid proteins show a variety of structural and functional similarities, it is unclear whether these similarities reflect descent from a common ancestor, convergent evolution, horizontal gene transfer among virus lineages, or acquisition of genes from host cells. Here, we report the discovery of a dozen new members of an emerging virus family, the Adomaviridae, that unite a papillomavirus/polyomavirus-like replicase gene with an adenovirus-like virion maturational protease. Adomaviruses were initially discovered in a lethal disease outbreak among endangered Japanese eels. New adomavirus genomes were found in additional commercially important fish species, such as tilapia, as well as in reptiles. The search for adomavirus sequences also revealed an additional candidate virus family, which we refer to as xenomaviruses, in mollusk datasets. Analysis of native adomavirus virions and expression of recombinant proteins showed that the virion structural proteins of adomaviruses are homologous to those of both adenoviruses and another emerging animal virus family called adintoviruses. The results pave the way toward development of vaccines against adomaviruses and suggest a framework that ties small DNA tumor viruses into a shared evolutionary history.Author SummaryIn contrast to cellular organisms, viruses do not encode any universally conserved genes. Even within a given family of viruses, the amino acid sequences encoded by homologous genes can diverge to the point of unrecognizability. Although members of an emerging virus family, the Adomaviridae, encode replicative DNA helicase proteins that are recognizably similar to those of polyomaviruses and papillomaviruses, the functions of other adomavirus genes have been difficult to identify. Using a combination of laboratory and bioinformatic approaches, we identify the adomavirus virion structural proteins. The results link adomavirus virion protein operons to those of other midsize non-enveloped DNA viruses, including adenoviruses and adintoviruses.

scholarly journals Expression and characterisation of the ryegrass mottle virus non-structural proteins

2010 ◽  
Vol 64 (5-6) ◽  
pp. 215-222
Author(s):  
Ina Baļķe ◽  
Gunta Resēviča ◽  
Dace Skrastiņa ◽  
Andris Zeltiņš
Keyword(s):  
Amino Acid ◽  
Expression System ◽  
Protein Structures ◽  
Amino Acid Sequences ◽  
Host Cells ◽  
Structural Proteins ◽  
Mottle Virus ◽  
Expression Vectors ◽  
Structural Protein ◽  
E Coli

Expression and characterisation of the ryegrass mottle virus non-structural proteins The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several E. coli host cells. For protease and RdRP, the best expression system containing pColdI vector and E. coli WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and E. coli BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.

scholarly journals Analysis of the Amino Acid Sequence Variation of the 67–72p Protein and the Structural Pili Proteins of Corynebacterium diphtheriae for their Suitability as Potential Vaccine Antigens

2019 ◽  
Vol 68 (2) ◽  
pp. 233-246
Author(s):  
KLAUDIA BRODZIK ◽  
KATARZYNA KRYSZTOPA-GRZYBOWSKA ◽  
MACIEJ POLAK ◽  
JAKUB LACH ◽  
DOMINIK STRAPAGIEL ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vaccine Development ◽  
Corynebacterium Diphtheriae ◽  
Amino Acid Sequences ◽  
Host Cells ◽  
Structural Proteins ◽  
Bioinformatics Analyses ◽  
Conserved Regions ◽  
Vaccine Antigens ◽  
Potential Vaccine

The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67–72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67–72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67–72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.

scholarly journals Yersiniophage ϕR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4093-4102 ◽  
Author(s):  
Saija Kiljunen ◽  
Kristo Hakala ◽  
Elise Pinta ◽  
Suvi Huttunen ◽  
Patrycja Pluta ◽  
...  
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Burst Size ◽  
Outer Core ◽  
International Committee ◽  
Amino Acid Sequences ◽  
Structural Proteins ◽  
Virulence Plasmid ◽  
O Antigen ◽  
Serotype O ◽  
Phage Receptor

Bacteriophage ϕR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulence-plasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9. Electron microscopy demonstrated that ϕR1-37 particles have an icosahedral head of 88 nm, a short neck of 10 nm, a long contractile tail of 236 nm, and tail fibres of at least 86 nm. This implies that the phage belongs to the order Caudovirales and the family Myoviridae in the ICTV (International Committee for Taxonomy of Viruses) classification. ϕR1-37 was found to have a lytic life cycle, with eclipse and latent periods of 40 and 50 min, respectively, and a burst size of ∼80 p.f.u. per infected cell. Restriction digestions and PFGE showed that the ϕR1-37 genome was dsDNA and ∼270 kb in size. Enzymically hydrolysed DNA was subjected to HPLC-MS/MS analysis, which demonstrated that the ϕR1-37 genome is composed of DNA in which thymidine (T) is >99 % replaced by deoxyuridine (dU). The only organisms known to have similar DNA are the Bacillus subtilis-specific bacteriophages PBS1 and PBS2. N-terminal amino acid sequences of four major structural proteins did not show any similarity to (viral) protein sequences in databases, indicating that close relatives of ϕR1-37 have not yet been characterized. Genes for two of the structural proteins, p24 and p46, were identified from the partially sequenced ϕR1-37 genome.

scholarly journals First Report of Apricot vein clearing-associated virus (AVCaV) infecting Prunus domestica revealed by High-Throughput Sequencing in Morocco

Plant Disease ◽  
2020 ◽  
Author(s):  
Rachid Tahzima ◽  
Radouane Qessaoui ◽  
Yoika Foucart ◽  
Sebastian Massart ◽  
Kris De Jonghe
Keyword(s):  
Fruit Trees ◽  
Plant Viruses ◽  
Amino Acid Sequences ◽  
Stone Fruit ◽  
Replicase Gene ◽  
Prunus Domestica ◽  
Prunus Species ◽  
First Report ◽  
Vein Clearing ◽  
The Impact

Plum (Prunus domestica L., Rosaceae) trees, like many stone fruit trees, are known to be infected by numerous plant viruses, predominantly as consequence of their clonal mode of propagation and perennial cultivation (Jelkmann and Eastwell, 2011). Apricot vein clearing-associated virus (AVCaV) is a member of the genus Prunevirus in the family Betaflexiviridae. AVCaV was first reported in Italy infecting apricot (P. armeniaca L.) associated with foliar vein clearing symptoms (Elbeaino et al. 2014). It has also been detected in various Prunus species, like plum, Japanese plum (P. salicina L.), sour cherry (P. cerasus L.), and Japanese apricot (P. mume L.), apricot and peach (P. persica L.) sourced from Asian and European countries (Marais et al. 2015), as well as in the ornamental Myrobolan plum (P. cerasifera L.) in Australia (Kinoti et al. 2017). In 2018, during the vegetative season, a survey was carried out in two different apricot and plum orchards in the southern region of Agdez (Agadir, Morocco) where stone fruit trees are grown. Five branches with leaves were sampled from three apricot and three plum trees of unknown cultivars, all asymptomatic. Total RNA was extracted from 100 mg plant tissue (leaves and cambial scrapping) using RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and separate samples (one per species) were used for library preparation (NEBNext Ultra RNA library kit; New England BioLabs, MA, USA), and sequencing (Illumina NextSeq v2, totRNA sequencing) at Admera Health (New Jersey, USA). All generated reads (6,756,881) from the plum sample were quality filtered and submitted to the VirusDetect pipeline (Zheng et al., 2017). The plum cDNA library, a total of 20 viral contigs (68-1928 bp) mapped to several AVCaV accessions in GenBank. A reference mapping (CLC Genomics Workbench 12, Qiagen, Denmark) was conducted against all four available AVCaV full genomes (KM507062-63, KY132099 and HG008921), revealing 100% coverage of the full sequence (8358 nt) with 97-98 % nucleotide (nt) identities (BLASTn). Analysis of the derived sequences allowed to identify the location of the four predicted ORFs i.e. (ORF1: 6066 nt/2,021 aa), (ORF2: 1383 nt/460 aa), (ORF3: 666 nt/221 aa) and (ORF4: 420 nt/139 aa), previously described for the AVCaV genome (Elbeaino et al. 2014). The amino acid sequences of the encoded proteins of AVCaV isolate from Morocco also shared 97-98% identities with the corresponding sequences of complete genome AVCaV isolates in GenBank. To confirm the detection of AVCaV in the three plum samples, specific RT-PCR primers (VC37657s: 5’-CCATAGCCACCCTTTTTCAA-3’ / VC28239a: 5’-GTCGTCAAGGGTCCAGTGAT-3’) (Elbeaino et al. 2014) were used and the expected 330 bp fragment from the replicase gene was amplified in all three samples and subsequently sequenced (MT980794-96). Sanger sequences were 100% identical to corresponding HTS derived sequence. This is the first report of AVCaV infecting plum in Africa. The incidence of AVCaV in Moroccan Prunus species is unknown. Plum trees from the surveyed orchards were also confirmed to be co-infected with little cherry virus 1 (LChV-1) using HTS. Further investigation is required to determine the impact of AVCaV on these asymptomatic plum trees and other stone fruits species.

O-β-GlcNAcylation, Chloroquine and 2-Hydroxybenzohydrazine May Hamper SARS-CoV-2 entry to Human via Inhibition of ACE2 Phosphorylation at Ser787 but Also Induce Disruption of Virus-ACE2 Binding

2020 ◽  
Author(s):  
Waqar Ahmad ◽  
Khadija Shabbiri ◽  
Nazarul Islam
Keyword(s):  
Human Body ◽  
Virus Entry ◽  
Critical Role ◽  
Virus Transmission ◽  
Binding Domain ◽  
Host Cells ◽  
Phosphoryl Group ◽  
Post Translational Modification ◽  
Virus Family ◽  
Downstream Signaling

The novel coronavirus COVID- 19 disease is extremely contagious and has been spread worldwide. First COVID-19 case was identified in December, 2019 and within three months, more than one million affected cases and over 65,000 deaths have been reported. SARS-coronavirus 2 (SARS-CoV-2) also known as 2019-nCoV is a causative agent of COVID-19 disease and belongs to the SARS CoV (Severe Acute Respiratory Syndrome corona virus) family. The SARS-CoV-2 enters the human body by binding its viral surface spike protein with the host angiotensin-converting enzyme 2 (ACE2) receptors and cause infection. To prevent the virus entry and its transmission in the human body, we focused on the two domains of ACE2: i) the N-terminal extracellular binding domain (18-740 residues) reported for coronavirus spike interaction, and ii) the C-terminal cytoplasmic region (762-805 residues) to prevent the virus transmission. Therefore, we proposed: i) inhibition of receptor binding domain (RBD) of SARS-CoV-2 and human ACE2 protein may prevent the virus entry to the host and ii) inhibition of phosphorylation at Ser-787 of ACE2 protein may prevent the transmission of the virus in the COVID-19 patients. In the past, the critical role of Ser 787 in human ACE2 protein has been experimentally verified in SARS-CoV transmission, that upon binding to the receptor, SARS- CoV induces CKII- mediated phosphorylation of ACE2 at Ser-787 that in-turn facilitate virus entry to host cells, followed by replication and activation of ACE2, initiates downstream signaling leading to lung fibrosis. Therefore, in this study, we have suggested post-translational modification (PTM) O-β-GlcNAcylation, and two compounds Chloroquine and 2-hydroxybenzohydrazine might share the common pathways to prevent the COVID-19 infection in human. The addition of O-β-GlcNAcylation at same or neighboring Ser/ Thr residues results in phosphorylation inhibition and a change in protein structural and functional confirmations. Thereby, using neural networking methods, we have identified Ser/ Thr residues in ACE2 that are potential sites for phosphorylation and / or O-β-GlcNAcylation. Molecular docking showed that UDP-GlcNAc has more binding affinity with Ser-787 than the phosphoryl group. Moreover, chloroquine and 2-hydroxybenzohydrazine also showed great potential to bind at Ser-787 that may result in inhibition of Ser-787 phosphorylation and downstream signaling. Furthermore, O-β-GlcNAcylation, chloroquine and 2-hydroxybenzohydrazine showed their high affinity at ACE2-SARS-CoV-2receptor binding domain that may prevent the entry of SARS-CoV-2 into human body. In conclusion, inhibition of human ACE2 phosphorylation at Ser-787 and ACE2-SARS-CoV-2 binding domain could be promising targets against SARS-CoV-2 infection.

scholarly journals Identification of porcine reproductive and respiratory syndrome virus ORF1a-encoded non-structural proteins in virus-infected cells

2012 ◽  
Vol 93 (4) ◽  
pp. 829-839 ◽  
Author(s):  
Yanhua Li ◽  
Ali Tas ◽  
Eric J. Snijder ◽  
Ying Fang
Keyword(s):  
Rna Synthesis ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antibodies ◽  
Structural Proteins ◽  
Respiratory Syndrome Virus ◽  
Replicase Gene ◽  
Future Studies ◽  
Swine Pathogen ◽  
Infected Cells

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase gene consists of two large ORFs, ORF1a and ORF1b, the latter of which is expressed by ribosomal frameshifting. The ORF1a-encoded part of the resulting replicase polyproteins (pp1a and pp1ab) is predicted to be processed proteolytically into ten non-structural proteins (nsps), known as nsp1–8, with both the nsp1 and nsp7 regions being cleaved internally (yielding nsp1α and nsp1β, and nsp7α and nsp7β, respectively). The experimental verification of these predictions depends strongly on the ability to identify individual cleavage products with specific antibodies. In this study, a panel of monoclonal and polyclonal antibodies was generated, which together were able to recognize eight ORF1a-encoded PRRSV nsps. Using these reagents, replicase cleavage products were detected in PRRSV-infected MARC-145 cells using a variety of immunoassays. By immunofluorescence microscopy, most nsps could be detected by 6 h post-infection. During the early stages of infection, nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8 co-localized in distinct punctate foci in the perinuclear region of the cell, which were determined to be the site of viral RNA synthesis by in situ labelling. Western blot and immunoprecipitation analysis identified most individual nsps and several long-lived processing intermediates (nsp3–4, nsp5–7, nsp5–8 and nsp3–8). The identification and subcellular localization of PRRSV nsps in virus-infected cells documented here provides a basis for the further structure–function studies. Thus, this PRRSV antibody panel will be an important tool for future studies on the replication and pathogenesis of this major swine pathogen.

Sign in / Sign up

Export Citation Format

Share Document