Rotavirus NSP1 localizes in the nucleus to disrupt PML nuclear bodies during infection

Abstract

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...

CRISPR-Csy4-Mediated Editing of Rotavirus Double-Stranded RNA Genome

“…The size of gs5 ∗ -HA-EGFP ∗ was too large to detect the edited segment by dsRNA gel migration ( Figure S6 A), but by Sanger sequencing decomposition ( Brinkman et al., 2014 ), the total editing efficiency was 9.6% and the only significant editing event was the in-frame Δ36 deletion ( Figure S6 B). Cytofluorimetric analysis ( Figure 4 B) and confocal fluorescence images ( Figures 4 C and 4D; Figure S6 C) of NSP1-EGFP spots ( Murphy and Arnold, 2019 ) showed 15% of EGFP-positive cells in rRV-infected MA-NSP5-Csy4 at 12 hpi. EGFP was detected only in cells sustaining viral replication (anti-NSP2) ( Figure 4 C) and was compromised upon inhibition of viroplasm formation by the proteasome inhibitor MG132 ( Contin et al., 2011 ; Figures 4 E and 4F; Figure S6 E).…”

Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission

“…Our data suggest that NSP1 likely facilitates virus replication independently of the IFN and inflammasome signaling and beyond one replication cycle. Interestingly, NSP1 has been shown to be localized to the nucleus during viral infection, and this relocalization disrupted the function of promyelocytic nuclear bodies, which was related to host stress responses ( 29 ). More recently, NSP1 has been linked to the inhibition of the transcription ( 30 ) but not the translation ( 14 ) of IRF1.…”

Re-Examining Rotavirus Innate Immune Evasion: Potential Applications of the Reverse Genetics System

“…In vivo attenuation could not be fully rescued in STAT1 knockout mice, suggesting a previously unknown role for NSP1 in IFN-independent signaling. NSP1 also seems to localize to the nucleus and disrupt promyelocytic nuclear bodies ( 211 ). Dissecting the complex activities and associated domains of the multifunctional NSP1 will be challenging, but key to a complete picture of RV innate immune evasion.…”