New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Laitinen, Olli H.; Svedin, Emma; Kapell, Sebastian; Hankaniemi, Minna M.; Larsson, Pär; Domsgen, Erna; Stone, Virginia M.; Määttä, Juha; Hyöty, Heikki; Hytönen, Vesa P.; Flodström‐Tullberg, Malin

doi:10.1016/j.jviromet.2018.02.001

Cited by 15 publications

(24 citation statements)

References 55 publications

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“…Purified calpain 1 (human) and 2 (rat) proteases were from Calbiochem. Purified viral proteases 2A and 3C were expressed in E. coli as described before [26]. Calpain inhibitor I (N-Acetyl-Leu-Leu-norleucinal) was from Roche (Basel, Switzerland).…”

Section: Reagentsmentioning

confidence: 99%

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Laajala

Hankaniemi

Määttä

et al. 2019

Viruses

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Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

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Section: Reagentsmentioning

confidence: 99%

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Laajala

Hankaniemi

Määttä

et al. 2019

Viruses

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“…The 'unbiased proteomic analysis' claimed in the article by Dunne et al to verify the presence of viral proteins in type 1 diabetic pancreases sounds interesting but is based on unpublished data only, and it is not clear whether the presence of VP1 correlates with IHC positivity or type 1 diabetes. A number of novel antibodies with demonstrated high specificity for structural [15,17] and non-structural [18] enterovirus proteins have been developed but, to date, none of these have been used to confirm the presence of enterovirus protein in the pancreas of individuals with type 1 diabetes.…”

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confidence: 99%

Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence

Skog

Klingel²,

Roivainen

et al. 2019

Diabetologia

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Abbreviations IHC Immunohistochemistry PKR Protein kinase R VP1 Viral protein 1To the Editor: We read with great interest the For Debate article by Dunne et al [1] and fully agree with the conclusion that intervention studies using a vaccine or antiviral drugs represent the way forward to finally establish whether enterovirus plays a role in type 1 diabetes. While we welcome and support such initiatives, we consider it crucial that funders investing in the huge vaccination trials required to prove or disprove the link are provided with solid scientific evidence. Also, in the risk-benefit analysis and ethical considerations, it is essential that the available evidence is viewed critically. Therefore, we feel compelled to point out some issues regarding the evidence cited in the article by Dunne et al.First, the sequence data of the enterovirus isolated by Dotta et al from an individual with recent-onset type 1 diabetes [2] shared 99% identity with the coxsackievirus B4 prototype strain JVB Benschoten, originally isolated in 1951, clearly indicating that it was in fact a laboratory contaminant. This is well known and non-controversial among virologists and others within the field [3-5] and we find it problematic that this paper is uncritically cited as supporting evidence for the presence of pancreatic enterovirus in type 1 diabetes.Second, in the Diabetes Virus Detection (DiViD) study, we detected enterovirus RNA with RT-PCR in pancreatic tissue samples from only one of the six patients with recent-onset type 1 diabetes [6]. This patient was also RT-PCR-positive for enterovirus RNA in peripheral blood mononuclear cells, suggesting that the positive signal could have come from infected circulating blood cells present in the pancreas, particularly given that this biopsy was collected as a surgical specimen and not perfused with preservation solution to remove blood, as is usually done during organ procurement for transplantation. The reported, and frequently cited, enterovirus RNA-positive samples from four out of six DiViD patients were from the culture medium of isolated islets. These islets were isolated from the tip of the pancreatic tail under non-sterile conditions in a research laboratory for the purpose of functional islet studies [7]. The enterovirus-negative islets from six non-diabetic organ donors [6], referred to by Dunne et al, were isolated in our Good Manufacturing Process (GMP) facility for the purpose of clinical islet transplantation and, therefore, these results cannot be used to rule out the possibility of viral contamination in the culture medium of islets isolated from the type 1 diabetic individuals. Also, the discrepancy in PCR positivity between tissue samples and the culture medium of isolated islets is difficult to explain by a difference in sensitivity [8], and the possibility that the positive signal was derived from contaminating viral sequences introduced during islet isolation or culture cannot be excluded. Other studies (not cited in the article by Dunne et al) have failed to find e...

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“…A good example is the recent study by Laitinen et al . , in which new monoclonal antibodies to the CVB‐encoded proteases 2Apro and 3Cpro were generated. These antibodies were able to detect the vast majority of EV‐B species in EV‐A‐, B‐ and C‐infected cells.…”

Section: Introductionmentioning

confidence: 99%

“…Investigators are focusing on producing more suitable reagents to specifically detect EVs, which might have important consequences for vaccine development. A good example is the recent study by Laitinen et al [14], in which new monoclonal antibodies to the CVB-encoded proteases 2Apro and 3Cpro were generated. These antibodies were able to detect the vast majority of EV-B species in EV-A-, B-and C-infected cells.…”

Section: Introductionmentioning

confidence: 99%

Enterovirus infection and type 1 diabetes: unraveling the crime scene

Rodríguez-Calvo

2018

Clinical and Experimental Immunology

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Summary Enteroviruses (EV) have been historically associated to type 1 diabetes. Definitive proof for their implication in disease development is lacking, but growing evidence suggests that they could be involved in beta cell destruction either directly by killing beta cells or indirectly by creating an exacerbated inflammatory response in the islets, capable of attracting autoreactive T cells to the ‘scene of the crime’. Epidemiological and serological studies have been associated with the appearance of islet autoimmunity and EV RNA has been detected in prospective studies. In addition, the EV capsid protein has been detected in the islets of recent‐onset type 1 diabetic donors, suggesting the existence of a low‐grade EV infection that could become persistent. Increasing evidence in the field shows that a ‘viral signature’ exists in type 1 diabetes and involves interferon responses that could be sustained during prolonged periods. These include the up‐regulation of markers such as protein kinase R (PKR), melanoma differentiation‐associated protein 5 (MDA5), retinoic acid inducible gene I (RIG‐I), myxovirus resistance protein (MxA) and human leukocyte antigen‐I (HLA‐I) and the release of chemokines able to attract immune cells to the islets leading to insulitis. In this scenario, the hyperexpression of HLA‐I molecules would promote antigen presentation to autoreactive T cells, favoring beta cell recognition and, ultimately, destruction. In this review, an overview is provided of the standing evidence that implicates EVs in beta cell ‘murder’, the time‐line of events is investigated from EV entry in the cell to beta cell death and possible accomplices are highlighted that might be involved in beta cell demise.

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New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Cited by 15 publications

References 55 publications

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence

Enterovirus infection and type 1 diabetes: unraveling the crime scene

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