Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. The roles of viral proteins in these processes have been studied mostly in vitro, by ectopic overexpression, or by surrogate infection systems, all of which have shortcomings. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro in the virus context. Viruses with an inactive 2A^pro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2A^pro is essential for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, suppressing the induction of the IFN response, and overcoming IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2A^pro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. Thus, 2A^pro plays a critical role in subverting antiviral responses and establishing a favorable environment to expedite enterovirus replication.

To understand viral infection and virus–host interactions, real-time, single-cell assays to track viral infection progression are essential. Many conventional assays sample large numbers of cells for single measurements, averaging out the cell-to-cell heterogeneity that is intrinsic to viral infection. Moreover, conventional assays often require cell fixation or lysis, limiting analysis to a single timepoint and masking the temporal and spatial dynamics of infection. We have developed virus infection real-time imaging (VIRIM), a method to visualize the translation of individual RNAs of viruses in real-time. The single-molecule and live-cell nature of VIRIM allows the examination of the earliest events of viral infection, when viral protein and RNA levels are still low, and allows study into the origins and consequences of cell-to-cell heterogeneity during virus infection. Here we provide a step-by-step description of the VIRIM assay, including a detailed procedure for designing, producing and validating the viruses required for VIRIM. In addition, we provide guidelines for generating the reporter cell line, performing the time-lapse imaging and analyzing the fluorescence microscopy data. Once established, a typical VIRIM experiment requires 2–5 days to complete.

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation.