Abstract
ABSTRACTInfection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-β treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-β-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures.
Highlights
All positive-strand RNA viruses of eukaryotes studied to date modify intracellular membranes into unique structures that presumably facilitate viral RNA synthesis
Our goal was to analyze whether the innate immune system responds to the formation of positive-strand RNA virus-induced replication organelles, and we hypothesized that such a response could be linked to the type I IFN signaling pathway, which has an important role in counteracting virus infections from their earliest stage onwards
When Equine arteritis virus (EAV)-infected HuH-7 cells were fixed by high-pressure freezing followed by freeze substitution (HPF-FS) and subsequently analyzed by electron microscopy (EM), double-membrane structures” (DMS) similar to those previously described upon EAV infection in other cell types were readily observed (Fig. 1C) [13, 23]
Summary
All positive-strand RNA viruses of eukaryotes studied to date modify intracellular membranes into unique structures that presumably facilitate viral RNA synthesis. Elaborate interactions between virus and host are believed to form the basis for the striking, virus-induced remodeling of specific cellular organelles in the infected cell [5,6,7,8] These replication organelles may consist of different substructures, such as spherules, tubules, convoluted membranes, paired membranes, or double-membrane vesicles. As for other positivestrand RNA viruses, the viral nonstructural proteins directly involved in RNA synthesis, such as the RNA-dependent RNA polymerase (RdRp), as well as the (presumed) double-stranded RNA (dsRNA) intermediates of viral RNA replication, colocalize with the membrane structures induced during EAV infection [1, 13, 21]. Only two reports have described such effects—in both cases on the membranous web formed during HCV replication [30, 31]; the underlying mechanisms remain to be fully characterized, and data for any other positive-strand RNA virus are lacking
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