Cell Biology and Anatomy
The replication in the mammalian cell cytoplasm of positive-strand RNA viruses and maturation of some DNA viruses takes place in association with intracellular membranes (the “membranous web”). The interferon-inducible human “myxovirus resistance protein A” (MxA) is a cytoplasmic dynamin-family large GTPase with broad-spectrum antiviral activity, which among its various actions, binds to and tubulates lipid membranes. Since 2002, MxA has been often stated to associate with “a subcompartment of the smooth endoplasmic reticulum” (ER) in uninfected cells, but without evaluation of this co-localization using structural proteins of the ER as markers. In a previous study this inference was evaluated using reticulon-4 (RTN4) and atlastin-3 (ATL3) as structural markers of the standard ER and immunostaining methods. MxA-HA transiently expressed in HEK293T, Cos7 or Huh7/ Huh7.5 cells was observed in the cytoplasm in a variety of phenotypic forms ranging from small and large endosomes to larger compact tubulo-reticular structures. The previous study showed that by immunofluorescence and immuno-electron microscopy methods, MxA-positive variably-sized endosomal and larger reticular structures were largely distinct from RTN4- and ATL3-based ER. In the present study this hypothesis was further tested using an alternative approach. Two-color fluorescence imaging was carried out on live and fixed Huh7 cells transiently expressing GFP-tagged MxA alone, or expressing MxA-GFP together with a mCherry-tagged ER marker RTN4-mCh. Imaging of the expression of MxA-GFP in Huh7 cells provided evidence for the association of MxA with cytoplasmic structures of different sizes and phenotypes, including rapidly dynamic endosomes and larger structures of low motility. In two-color fluorescence imaging assays these MxA-GFP structures were distinct from RTN4-mCh. The new data, together with the earlier immunofluorescence and immuno-electron microscopy observations, call for a reinterpretation of studies over the last 15 years of MxA cell biology and antiviral mechanisms. We suggest that MxA generated an intracellular membrane compartment distinct from the standard RTN4-based ER. This MxA-reticulum likely efficiently sequesters viral proteins to generate an antiviral phenotype.
Davis, Deodate; Yuan, Huijuan; Liang, Feng-Xia; Petzold, Chris; Dancel-Manning, Kristen; and Sehgal, Pravin B., "Cytoplasmic MxA-GFP Structures Are Distinct From The Standard Reticulon 4-Based Endoplasmic Reticulum" (2017). NYMC Faculty Posters. 12.