Antiviral responses are shaped by heterogeneity in viral replication dynamics

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

General structure of the Rap2 proteins and constructs used in this study.

<p>A: Domain structure of Rap2 isoforms. Proteins differ most extensively in the C-terminal hypervariable region (HVR), which comprises a linker and anchor region. Cysteine residues in the anchor region (highlighted in red) are posttranslationally subjected to CAAX box isoprenylation and dynamic cysteine palmitoylation. B: Schematic structure of various Rap2 constructs used in this study.</p

Localization of Rap2 isoforms in polarized Ls174T-W4 cells.

<p>A: GFP-tagged Rap2 isoform were cotransfected with the actin marker Lifeact-Ruby in W4 cells and imaged in unpolarized and polarized (i.e. doxycycline-stimulated) cells. B: GFP-Rap2 localization compared with the recycling endosomal marker dsRed-Rab11 in polarized W4 cells. Asterisks indicate the apical aspect as judged by polarized Rab11 distribution. C: Localization of the isolated hypervariable regions of the Rap2 isoforms. Profile plots show normalized fluorescence intensities over the indicated line scans. DOX: doxycyline.</p

CAAX-box swopped Rap2A and Rap2B mutants function indistinguishable from the wild type Rap2 proteins.

<p>A: Images of Rap2A depleted W4 cells expressing GFP-tagged CAAX-swopped Rap2 constructs and Lifeact-Ruby. B: Quantification of brush border formation in Rap2A-depleted W4 cells in which GFP-tagged CAAX-box mutant Rap2A and Rap2B were expressed. (Total counts ∼150 cells per condition) *p<0,05 using paired samples t-test. C: Western blot of lysates from a rescue experiment probed with anti-Rap2 antibody and anti-αTubulin as a loading control. shCTRL: non-targeting control short hairpin, EV: empty vector.</p

Mechanisms of Isoform Specific Rap2 Signaling during Enterocytic Brush Border Formation

<div><p>Brush border formation during polarity establishment of intestinal epithelial cells is uniquely governed by the Rap2A GTPase, despite expression of the other highly similar Rap2 isoforms (Rap2B and Rap2C). We investigated the mechanisms of this remarkable specificity and found that Rap2C is spatially segregated from Rap2A signaling as it is not enriched at the apical membrane after polarization. In contrast, both Rap2A and Rap2B are similarly located at Rab11 positive apical recycling endosomes and inside the brush border. However, although Rap2B localizes similarly it is not equally activated as Rap2A during brush border formation. We reveal that the C-terminal hypervariable region allows selective activation of Rap2A, yet this selectivity does not originate from the known differential lipid modifications of this region. In conclusion, we demonstrate that Rap2 specificity during brush border formation is determined by two distinct mechanisms involving segregated localization and selective activation.</p></div

Constitutively activated Rap2B(V12) or a chimeric Rap2B(A-HVR) mutant can rescue brush border formation in Rap2A depleted Ls174T-W4 cells.

<p>A: Images of Rap2A depleted polarized W4 cells expressing various GFP-tagged Rap2 constructs and LifeAct-Ruby. B: Quantification of brush border formation in GFP-positive cells in three independent experiments. (total counts ∼150 cells per condition). *p<0,05 using paired samples t-test. C: Western blot of lysates from a rescue experiment probed with anti-Rap2 antibody and anti-αTubulin as a loading control. D: GTP-Rap pulldown from unpolarized and polarized W4 cells expressing V5-Rap2A, V5-Rap2B or V5-Rap2B(A-HVR). Results from three independent experiments were quantified and averages were expressed as ratio of GTP-Rap2 vs. total Rap2 relative to unstimulated Rap2A. E: Image of Rap2A-depleted W4 cell expressing GFP-Rap2A(B-HVR) and LifeAct-Ruby. F: Quantification of brush border formation in Rap2A depleted W4 cells in which GFP or GFP-Rap2A(B-HVR) was introduced. (Total counts ∼300 cells per condition). *p<0,05 using paired samples t-test. shCTRL: non-targeting control short hairpin, EV: empty vector, DOX: doxycycline, GTP-Rap P.D.: GTP-bound Rap pulldown.</p

A Tuba/Cdc42/Par6A complex is required to ensure singularity in apical domain formation during enterocyte polarization.

Apico-basal polarity establishment is a seminal process in tissue morphogenesis. To function properly it is often imperative that epithelial cells limit apical membrane formation to a single domain. We previously demonstrated that signaling by the small GTPase Cdc42, together with its guanine nucleotide exchange factor (GEF) Tuba, is required to prevent the formation of multiple apical domains in polarized Ls174T:W4 cells, a single cell model for enterocyte polarization. To further chart the molecular signaling mechanisms that safeguard singularity during enterocyte polarization we generated knockout cells for the Cdc42 effector protein Par6A. Par6A loss results in the formation of multiple apical domains, similar to loss of Cdc42. In Par6A knockout cells, we find that active Cdc42 is more mobile at the apical membrane compared to control cells and that wild type Cdc42 is more diffusely localized throughout the cell, indicating that Par6A is required to restrict Cdc42 signaling. Par6A, Cdc42 and its GEF Tuba bind in a co-immunoprecipitation experiment and they partially colocalize at the apical membrane in polarized Ls174T:W4 cells, suggesting the formation of a trimeric complex. Indeed, in a rescue experiment using Par6A mutants, we show that the ability to establish this trimeric complex correlates with the ability to restore singularity in Par6A knockout cells. Together, these experiments therefore indicate that a Tuba/Cdc42/Par6A complex is required to ensure the formation of a single apical domain during enterocyte polarization

Translation and Replication Dynamics of Single RNA Viruses

RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses