word count: 211 14 Importance word count: 131 15 Text word count: 6,050 16 17 ABSTRACT 20The rotavirus nonstructural protein 1 (NSP1) antagonizes interferon (IFN) induction in 21 infected host cells. The primary function of NSP1 is thought to be degradation of interferon 22 regulatory factors (IRFs) and beta-transducin repeat-containing protein (β-TrCP) in the 23 cytoplasm to inhibit IFN induction. Here, we report that NSP1 localizes to the cytoplasm and 24 nucleus and disrupts promyelocytic (PML) nuclear bodies (NB) in the nucleus during infection. 25Nuclear localization of NSP1 did not require an intact C terminus, suggesting NSP1 has a novel 26 function in the nucleus independent of degradation of IRFs or β-TrCP. NSP1 expression either 27 led to a reduction in PML NB number or a change in PML NB morphology from sphere-shaped 28 foci to oblong-shaped structures, depending on the virus strain. Additionally, infection was not 29 affected when cells lack PML NB, suggesting that rotavirus does not require PML for replication 30 in highly permissive cell types. PML was not essential for nuclear localization of NSP1, but PML 31 was required for NSP1 nuclear focus formation. PML NBs play an important role in many 32 cellular functions that include IFN induction and host stress responses. This is the first report 33 that rotavirus, a cytoplasmically replicating virus, encodes a viral protein that localizes to the 34 nucleus during infection, and may suggest a new function of NSP1 in the nucleus. 35 36 IMPORTANCE 37Rotavirus causes severe gastroenteritis in young children and leads to over 200,000 38 deaths per year. Rotavirus is a cytoplasmically replicating virus, and must find ways to avoid or 39 actively inhibit host antiviral responses to efficiently replicate. The nonstructural protein NSP1 is 40 known to inhibit IFN induction by promoting degradation of host proteins in the cytoplasm of 41 infected cells. Here, we demonstrate that NSP1 also localizes to the nucleus of infected cells, 42 specifically to PML NB. NSP1 causes a disruption of PML NB, which may serve as an additional 43 mechanism of IFN inhibition or interfere with other nuclear processes to promote viral packaged inside a multi-layered, non-enveloped viral particle (1). Most steps of the rotavirus 50 replication cycle take place in the cytoplasm of infected cells in replication centers known as 51 viroplasms, and final assembly takes place via a process of budding through the endoplasmic 52 reticulum (ER) (2). Although the virus relies on its host to replicate efficiently, it must also find 53 ways to avoid host antiviral responses. As with many viruses, rotaviruses target several different 54 steps of the type I interferon (IFN) response in order to prevent the expression or activity of host 55 antiviral proteins. For instance, the viral protein VP3 functions within the innermost layer of the 56 viral particle as the capping enzyme, but also works within the infected cells to cleave 2'-5'-57 oligoadenylates to inhibit the activation of ribonuclease L...