scholarly journals Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 896 ◽  
Author(s):  
Shaochuan Zhou ◽  
Xinna Ge ◽  
Can Kong ◽  
Teng Liu ◽  
Aijing Liu ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Biochemical Properties ◽  
In Vitro Assays ◽  
Respiratory Syndrome Virus ◽  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Cleavage Activity ◽  
Tnf Α ◽  
Cis And Trans

The papain-like cysteine protease 2 (PLP2) within the N-terminus of the porcine reproductive and respiratory syndrome virus (PRRSV) nsp2 replicase protein specifies a deubiquitinating enzyme (DUB), but its biochemical properties and the role in infection have remained poorly defined. By using in vitro assays, we found that the purified PLP2 could efficiently cleave K63 and K48 linked polyubiquitin chains Ub3-7 in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. The subsequent mutagenesis analyses revealed that the requirement for PLP2 DUB activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., D91R, D85R, etc.) that largely ablated the DUB function also blocked the cis- but not trans-proteolytic cleavage of nsp2/3 polyprotein. Moreover, the analyses identified key mutations that could differentiate DUB from PLP2 cis- and trans-cleavage activities. Further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the DUB activity were all lethal to the virus, (ii) a point mutation T88G that selectively blocked the cis-cleavage activity of PLP2 did not affect viral viability in cell culture, and (iii) an E90Q mutation that did not affect either of the PLP2 activities led to rescue of WT-like virus but displayed significantly reduced ability to induce TNF-α production. Our findings support the possibility that the PLP2 DUB activity, but not cis-cleavage activity, is essential for PRRSV replication. The data also establish a strong link of nsp2 to pro-inflammatory cytokine induction during infection that operates in a manner independent of PLP2 DUB activity.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

scholarly journals A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection

2015 ◽  
Vol 89 (23) ◽  
pp. 12070-12083 ◽  
Author(s):  
Hiep L. X. Vu ◽  
Fangrui Ma ◽  
William W. Laegreid ◽  
Asit K. Pattnaik ◽  
David Steffen ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Vaccine Coverage ◽  
Rna Virus ◽  
Respiratory Syndrome Virus ◽  
Full Genome ◽  
Genome Sequences ◽  
Prrsv Strain ◽  
Heterologous Protection

ABSTRACTCurrent vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, bothin vitroandin vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine.IMPORTANCEThe extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called “centralized” sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

scholarly journals In Vitro Antioxidant Activity of Crude Extracts of Harpagophytum Zeyheri and Their Anti-Inflammatory and Cytotoxicity Activity Compared With Diclofenac.

2021 ◽  
Author(s):  
Sibonokuhle F. Ncube ◽  
Lyndy J. McGaw ◽  
Emmanuel Mfotie Njoya ◽  
Hilton G.T. Ndagurwa ◽  
Peter J. Mundy ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Ethyl Acetate ◽  
Inflammatory Activity ◽  
In Vitro Assays ◽  
Cytotoxicity Activity ◽  
Tnf Α ◽  
Low Toxicity ◽  
Anti Inflammatory ◽  
Ethyl Acetate Extracts

Abstract Background This study evaluated the in vitro antioxidant activity and comparison of anti-inflammatory and cytotoxic activity of Harpagopytum zeyheri with diclofenac. Methods In vitro assays were conducted using water, ethanol and ethyl acetate extracts of H.zeyheri. The antioxidant activity was evaluated using the 2,2'-diphenyl-1-picrylhydrazy (DPPH) and 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)assays. The anti-inflammatory activity was determined by measuring the inhibition of nitric oxide (NO) on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages as well as cytokine (TNF-α and IL-10) expression on LPS-induced U937 human macrophages. For cytotoxicity, cell viability was determined using the 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results The ethyl acetate extract had the lowest IC50 values in the DPPH (5.91µg/ml) and ABTS (20.5µg/ml) assay compared to other extracts. Furthermore, the ethyl acetate extracts effectively inhibited NO and TNF-α and proved to be comparable to diclofenac at some concentrations. All extracts of H. zeyheri displayed dose dependent activity and were associated with low levels of human-IL-10 expression compared to quercetin. Furthermore, all extracts displayed low toxicity relative to diclofenac. Conclusions These findings show that H. zeyheri has significant antioxidant activity. Additionally, similarities exist in inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac.

scholarly journals Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

2021 ◽  
Vol 22 (16) ◽  
pp. 9056
Author(s):  
Ada Połońska ◽  
Katarzyna Jasieniecka-Gazarkiewicz ◽  
Lingjie You ◽  
Xiahui Hao ◽  
Sylwia Klińska ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Polyunsaturated Fatty Acids ◽  
Ph Value ◽  
Biochemical Properties ◽  
In Vitro Assays ◽  
Efficient Production ◽  
Genome Database ◽  
Response To Temperature

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).

scholarly journals Characterization of OTUB1 activation and inhibition by different E2 enzymes

2019 ◽  
Author(s):  
Lauren T. Que ◽  
Marie E. Morrow ◽  
Cynthia Wolberger
Keyword(s):  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Kinetics And Thermodynamics ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

scholarly journals In vitro antioxidant activity of crude extracts of Harpagophytum zeyheri and their anti-inflammatory and cytotoxicity activity compared with diclofenac

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sibonokuhle F. Ncube ◽  
Lyndy J. McGaw ◽  
Emmanuel Mfotie Njoya ◽  
Hilton G. T. Ndagurwa ◽  
Peter J. Mundy ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Ethyl Acetate ◽  
Inflammatory Activity ◽  
In Vitro Assays ◽  
Tnf Α ◽  
Low Toxicity ◽  
Anti Inflammatory ◽  
Ethyl Acetate Extracts ◽  
In Vitro Antioxidant Activity

Abstract Background This study evaluated the in vitro antioxidant activity and comparison of anti-inflammatory and cytotoxic activity of Harpagopytum zeyheri with diclofenac. Methods In vitro assays were conducted using water, ethanol, and ethyl acetate extracts of H.zeyheri. The antioxidant activity was evaluated using the 2,2′-diphenyl-1-picrylhydrazy (DPPH) and 2,2′- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The anti-inflammatory activity was determined by measuring the inhibition of nitric oxide (NO) on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages as well as cytokine (TNF-α and IL-10) expression on LPS-induced U937 human macrophages. For cytotoxicity, cell viability was determined using the 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results The ethyl acetate extract had the lowest IC50 values in the DPPH (5.91 μg/ml) and ABTS (20.5 μg/ml) assay compared to other extracts. Furthermore, the ethyl acetate extracts effectively inhibited NO and TNF-α and proved to be comparable to diclofenac at some concentrations. All extracts of H. zeyheri displayed dose-dependent activity and were associated with low levels of human-IL-10 expression compared to quercetin. Furthermore, all extracts displayed low toxicity relative to diclofenac. Conclusions These findings show that H. zeyheri has significant antioxidant activity. Additionally, similarities exist in the inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac.

scholarly journals Targeted mutations in a highly conserved motif of the nsp1β protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus

2013 ◽  
Vol 94 (9) ◽  
pp. 1972-1983 ◽  
Author(s):  
Yanhua Li ◽  
Longchao Zhu ◽  
Steven R. Lawson ◽  
Ying Fang
Keyword(s):  
Gene Expression ◽  
Reverse Genetics ◽  
Innate Immune ◽  
Respiratory Syndrome Virus ◽  
Structural Protein ◽  
Virus Growth ◽  
Conserved Motif ◽  
Immune Genes ◽  
Infected Cells

Non-structural protein 1β (nsp1β) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a papain-like cysteine protease (PLPβ) domain and has been identified as the main viral protein antagonizing the host innate immune response. In this study, nsp1β was determined to suppress the expression of reporter genes as well as to suppress ‘self-expression’ in transfected cells, and this activity appeared to be associated with its interferon (IFN) antagonist function. To knock down the effect of nsp1β on IFN activity, a panel of site-specific mutations in nsp1β was analysed. Double mutations K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV) targeting a highly conserved motif of nsp1β, GKYLQRRLQ (in bold), impaired the ability of nsp1β to suppress IFN-β and reporter gene expression, as well as to suppress ‘self-expression’ in vitro. Subsequently, viable recombinant viruses vSD01-08-K130A/R134A and vSD95-21-K124A/R128A, containing double mutations in the GKYLQRRLQ motif were generated using reverse genetics. In comparison with WT viruses, these nsp1β mutants showed impaired growth ability in infected cells, but the PLPβ cleavage function was not directly affected. The expression of selected innate immune genes was determined in vSD95-21-K124A/R128A mutant-infected cells. The results consistently showed that gene expression levels of IFN-α, IFN-β and IFN-stimulated gene 15 were upregulated in cells that were infected with the vSD95-21-K124A/R128A compared with that of WT virus. These data suggest that PRRSV nsp1β may selectively suppress cellular gene expression, including expression of genes involved in the host innate immune function. Modifying the key residues in the highly conserved GKYLQRRLQ motif could attenuate virus growth and improve the cellular innate immune responses.

scholarly journals Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Xufang Deng ◽  
Robert C. Mettelman ◽  
Amornrat O’Brien ◽  
John A. Thompson ◽  
Timothy E. O’Brien ◽  
...  
Keyword(s):  
Viral Replication ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Polyubiquitin Chains ◽  
Host Innate Immune Response ◽  
Necessary And Sufficient

ABSTRACTAnalysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferonin vitroand are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCECoronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

Quantitative Association Tests of Immune Responses to Antigens ofMycobacterium Tuberculosis: A Study of Twins in West Africa

Twin Research ◽  
2004 ◽  
Vol 7 (6) ◽  
pp. 578-588 ◽  
Author(s):  
Amanda Wiart ◽  
Annette Jepson ◽  
Winston Banya ◽  
Steve Bennett ◽  
Hilton Whittle ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Therapeutic Interventions ◽  
In Vitro Assays ◽  
Peptide Antigens ◽  
Tnf Α ◽  
Genetic Mechanisms ◽  
Host Genetic

AbstractThere is now considerable evidence that host genetic factors are important in determining the outcome of infection withMycobacterium tuberculosis(MTB). The aim of this study was to assess the role of several candidate genes in the variation observed in the immune responses to MTB antigens. In-vitro assays of T-cell proliferation, an in-vivo intradermal delayed hypersensitivity response; cytokine and antibody secretions to several mycobacterial peptide antigens were assessed in healthy, but exposed, West African twins. Candidate gene polymorphisms were typed in theNRAMP1,Vitamin D receptor,IL10,IL4,IL4 receptorandCTLA-4genes. Variants of the lociIL10(−1082 G/A),CTLA-4(49 A/G) and theIL4 receptor(128 A/G) showed significant associations with immune responses to several antigens. T-cell proliferative responses and antibody responses were reduced, TNF-α responses were increased for subjects with theCTLA-4G allele. The T-cell proliferative responses of subjects withIL10GA and GG genotypes differed significantly.IL4 receptorAG and GG genotypes also showed significant differences in their T-cell proliferative responses to MTB antigens. These results yield a greater understanding of the genetic mechanisms that underlie the immune responses in tuberculosis and have implications for the design of therapeutic interventions.

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