key: cord-0866518-sm5o0g1c authors: Eichwald, Catherine; Ackermann, Mathias; Fraefel, Cornel title: Mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components. date: 2020-08-04 journal: Virology DOI: 10.1016/j.virol.2020.07.008 sha: 8d474f1e06c53fd6542992ccc713f1b1ed45bc02 doc_id: 866518 cord_uid: sm5o0g1c Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV μ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a μ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-μ2 and μNS. Thus, μ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs. Here, specific µ2 punctae observed in filamentous VFs are investigated concerning their 89 ability to co-localize with other reovirus proteins and host elements. Our study shows that µ2 90 punctae in VFs co-localize with γ-tubulin, are resistant to nocodazole, and permit MT 91 emergence, common features for MTOCs. Moreover, using the VFLS model, we found that 92 specific µ2/µNS ratios that support filamentous morphology relocalize γ-tubulin and centrin to 93 foci within the VFLS. Such association is obliterated upon MT overexpression. 94 Immunofluorescence microscopy of reovirus T1L infected cells at 12 hpi, revealed µ2 97 punctae inside the filamentous VFs (Fig 1A) . The punctae co-localized neither with other viral 98 proteins (µNS, σNS, λ2, σ3, µ1) (Fig 1B-E (Fig 2D and E) . Co-staining for µ2 and α-tubulin, however, 100 showed bundles of MTs extending from the punctae, suggesting that the punctae may have a role 101 as MTOCs (Fig. 2C) . Indeed, co-staining for µ2 and γ-tubulin, a conventional marker for 102 centrosomes and other MTOCs (Roostalu and Surrey, 2017) , showed µ2 and γ-tubulin co-103 localizing in the punctae as denoted by immunofluorescence photomicrograph and profile 104 intensities of the linear region of interest (LROI) (Fig 2B) . Importantly, nocodazole treatment, 105 which is a well-known MT-depolymerizing agent, failed to disrupt the punctae, which remained 106 positive for both µ2 and γ-tubulin (Fig 3A and B) . Our results show that γ-tubulin localization is 107 intensified in µ2 punctae upon nocodazole treatment (Fig 3D) , consistent with the fact that 108 MTOCs are nocodazole resistant (Rogalski and Singer, 1984) . Reovirus protein µNS is mainly 109 dispersed from punctae when cells are treated with nocodazole (Fig 3A) , suggesting a mild or no 110 J o u r n a l P r e -p r o o f role in µ2 punctae formation. As expected, MT bundles depolymerized upon nocodazole 111 treatment (Fig 3C) Mainou et al., 2013) . Nocodazole was then removed from the medium, allowing MTs to re-117 polymerize for 0, 5, 15, 30, or 60 min before methanol-fixation (Fig 4A) . Immediately after 118 nocodazole removal (i.e., at 0 min), µ2-punctae were observed in the VFs while filamentous µ2 119 and MTs were not. However, within only 15 min after nocodazole removal, polymerizing MTs 120 with associated µ2 were observed extending from the punctae (Fig 4B, C, and D) . 5 and 6 , first, fourth, and fifth rows) mainly localized in MTOCs. In 134 contrast, γ-tubulin and centrin localized to defined punctae in VFLS reconstituted with µ2/µNS 135 transfection ratios of 2:1 and 2:2 ( Figures 5 and 6, second and third rows) . To evaluate if γ-136 tubulin and centrin punctae increment in number and redistribute to VFLSs generated with 137 different µ2/µNS ratios, we first quantified cells with more than two γ-tubulin punctae. As 138 denoted in Figure 7A , a significantly higher percentage of cells containing >2 MTOCs is 139 observed at 2:1 and 2:2 transfection ratios of µ2(T1L)/µNS when compared to the other ratios. 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