Exploration of IRES Elements within the ORF of the Coxsackievirus B3 Genome.

Objective: This study aimed to identify internal ribosome entry sites (IRESs) in the open reading frame (ORF) of the Coxsackievirus B3 (CVB3) genome. Methods: The sequences of P1, P2, or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin (ATG) to the end of the P1, P2, or P3 gene were inserted into the pEGFP-N1 vector. After transfection, possible IRES-dependent green fluorescent protein (GFP)-fused proteins were detected by anti-GFP western blotting. The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors, in which the potential IRESs were determined according to the Fluc/Rluc activity ratio. Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR. Results: After transfection of full length or truncated sequences of the P1, P2, or P3 plasmids, six GFP-fused protein bands in P1, six bands in P2 and nine bands in P3 were detected through western blotting. Two IRESs in VP2 (1461-1646 nt) and VP1 (2784-2983 nt) of P1; one IRES in 2C (4119-4564 nt) of P2; and two IRESs in 3C (5634-5834 nt) and 3D (6870-7087 nt) of P3 were identified according to Fluc/Rluc activity ratio. The cryptic promoter was also excluded by RT-qPCR. Conclusion: Five IRESs are present in the CVB3 coding region.

Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress.

OBJECTIVE: Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress. METHODS: In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3). RESULTS: CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells. CONCLUSION: CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

Identification of the internal ribosome entry sites (IRES) of prion protein gene.

Many studies demonstrated that there are several type bands of prion protein in cells. However, the formation of different prion protein bands is elusive. After several low molecular weight bands of prion protein appeared in SMB-S15 cells infected with scrapie agent Chandler, we think that IRES-dependent translation mechanism induced by prion is involved in the formation of prion protein bands. Then we designed a series of pPrP-GFP fusing plasmids and bicistronic plasmids to identify the IRES sites of prion protein gene and found 3 IRES sites inside of PrP mRNA. We also demonstrated that cap-independent translation of PrP was associated with the ER stress through Tunicamycin treatment. We still found that only IRE1 and PERK pathway regulated the IRES-dependent translation of PrP in this study. Our results indicated, we found that PrP gene had an IRES-dependent translation initiation mechanism and we successfully identified the IRESs inside of the prion protein gene.

Viral Contamination Source in Clinical Microbiology Laboratory.

To understand the potential causes of laboratory-acquired infections and to provide possible solutions that would protect laboratory personnel, samples from a viral laboratory were screened to determine the main sources of contamination with six subtypes of Rhinovirus. Rhinovirus contamination was found in the gloves, cuffs of protective wear, inner surface of biological safety cabinet (BSC) windows, and trash handles. Remarkably, high contamination was found on the inner walls of the centrifuge and the inner surface of centrifuge tube casing in the rotor. Spilling infectious medium on the surface of centrifuge tubes was found to contribute to contamination of centrifuge surfaces. Exposure to sodium hypochlorite containing no less than 0.2 g/L available chlorine decontaminated the surface of the centrifuge tubes from Rhinovirus after 2 min.

Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases.