Rhinovirus 2A is the predominant protease responsible for instigating the early block to gene expression encountered in infected cells

Abstract

1Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for 2 viral polyprotein processing. Both proteases also suppress host gene expression by 3 inhibiting mRNA transcription, nuclear export and cap-dependent translation. 4However, the relative contribution that each makes in achieving this goal remains 5 Methods 83Cell lines and reagents 84 293T cells were maintained at 37°C in a 5% CO2 incubator with DMEM (Invitrogen, 85Fisher Scientific UK Ltd, Loughborough, UK) supplemented with 10% foetal bovine 86 serum, 100 U/ml penicillin, 100 µg/ml streptomycin. Experimental treatment of cells 87 included incubation in medium supplemented with 200 µg/ml cycloheximide (Sigma, 88 Gillingham, UK) and/or 5 µg/ml actinomycin D (Cayman Chemical Cambridge 89Bioscience Ltd, Cambridge, UK). 90 91 DNA constructs 92Generation of some constructs relied on gene synthesis (Invitrogen, Fisher Scientific 93 UK Ltd, Loughborough, UK). In instances where this was the case, sequences ordered 94 were codon optimized for mammalian expression and then manually adjusted to 95 minimize the existence of unwanted splice donor and splice acceptor sites using the 96 online programmes HSF3[25] and the NetGene2 server [26]. The initial plasmid 97 expressing GFP, VSV-tagged 2A and HA-tagged 3C from HRV16 was generated by 98 cloning a single synthetic DNA fragment into an in-house dual promoter plasmid via 99EcoRI and SalI restriction sites to generate pCIPEP-A16 TAG (+/+) (see Fig. S1 for 100 sequence). Synthesized DNAs encoding for HRV-B4 and HRV-C2 proteases (Table 101 S1) were exchanged with their respective counterparts in pCIPEP-A16 TAG (+/+) using 102ClaI and SbfI (2A) and BsiWI and SalI (3C) restriction sites. Inactivation of the 103 proteases involved a two-step PCR approach, which introduced a Cys>Ala mutation 104 in the active site of both 2A and 3C. A similar PCR-based strategy was used to 105 generate constructs expressing tag-free proteases and a 3C protease with a c-Myc 106 nuclear localization signal (PAAKRVKLD) fused directly to its C-terminus. Primer 107 sequences are available on request. 108The synthetic DNA encoding for the Thioredoxin-Nanoluciferase(Nluc)PEST 109 fusion protein linked by a peptide derived from translating the hepatitis delta virus 110 (HdV) ribozyme sequence (HdV WT Nluc; Fig. S2) was initially cloned into pCDNA3.