The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

Martínez-Álvarez, Laura; Piña-Vázquez, Carolina; Zarco, Wilbert; Padilla-Noriega, Luis

doi:10.1590/s0074-0276108042013005

Cited by 8 publications

(8 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4C). These observations are consistent with those of previous studies (25)(26)(27) and indicate that the distribution of rsSA11-GFP11 NSP1 is the same as that of native NSP1.…”

Section: Resultssupporting

confidence: 93%

Entirely plasmid-based reverse genetics system for rotaviruses

Kanai

Komoto

Kawagishi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

181

271

View full text Add to dashboard Cite

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

show abstract

“…4C). These observations are consistent with those of previous studies (25)(26)(27) and indicate that the distribution of rsSA11-GFP11 NSP1 is the same as that of native NSP1.…”

Section: Resultssupporting

confidence: 93%

Entirely plasmid-based reverse genetics system for rotaviruses

Kanai

Komoto

Kawagishi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

181

271

View full text Add to dashboard Cite

show abstract

“…Through insertion of reporter genes into the segment 5 dsRNA, recombinant RVAs that express reporter fluorescent proteins (FPs) (e.g., mCherry and enhanced green FP [EGFP]) have been produced (6,9,12); these FP-RVAs are important tools for studying virus replication and pathogenesis via live-cell imaging and other fluorescence-based approaches. Unfortunately, the segment 5 product NSP1 is expressed at low levels in infected cells and is subject to proteasomal degradation, making FP-fused NSP1 proteins less than ideal probes of RVA biology (13). Moreover, FP genes were inserted into the segment 5 dsRNA in such a way as to alter the NSP1 ORF, likely compromising the protein's function as an interferon antagonist (14-15) and thus affecting the biological properties of the virus.…”

mentioning

confidence: 99%

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

Philip

Perry

Eaton

et al. 2019

J Virol

View full text Add to dashboard Cite

Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo. Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA. IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

show abstract

“…Previously published studies have concluded that NSP1 localizes to the cytoplasm of infected cells, based on immunofluorescence imaging (17–19). Re-examination of images from these studies indicates that NSP1 from SA11-5N, UK, UK variant brvA, and RRV did localize to the nucleus, sometimes as punctate spots, in addition to its cytoplasmic localization.…”

Section: Discussionmentioning

confidence: 99%

“…Other cellular targets of NSP1 have also been identified, all of which appear to be targeted for degradation by the proteasome (13–16). NSP1 has been shown to localize diffusely throughout the cytoplasm of infected cells, but appears to be excluded from the viroplasms (17–19). Recent studies using screening approaches to identify host proteins that associate with NSP1 have found evidence for nuclear proteins in NSP1 pull down samples, but much of this data not been validated or investigated in detail (20, 21).…”

Section: Introductionmentioning

confidence: 99%

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

Murphy

Arnold

2019

Preprint

View full text Add to dashboard Cite

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...

show abstract

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

Cited by 8 publications

References 27 publications

Entirely plasmid-based reverse genetics system for rotaviruses

Entirely plasmid-based reverse genetics system for rotaviruses

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

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

Contact Info

Product

Resources

About