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

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

rotavirus; gene expression; interferons type 1; proteasome endopeptidase complex; ubiquitin protein ligases

Authorship

Laura Martínez-Álvarez

Carolina Piña-Vázquez

Wilbert Zarco

Luis Padilla-Noriega ⁺+ Corresponding author: lpadilla@unam.mx

+ Corresponding author: lpadilla@unam.mx

SCIMAGO INSTITUTIONS RANKINGS

Universidad Nacional Autónoma de México, Instituto de Investigaciones Biomédicas, Mexico, DFUniversidad Nacional Autónoma de MéxicoMexico, DFUniversidad Nacional Autónoma de México, Instituto de Investigaciones Biomédicas, Mexico, DF

Figures

Figures (5)

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Fig. 1
intracellular localisation of rhesus rotavirus (RRV) non-structural protein 1 (NSP1). A: recombinant NSP1 localises to the cytoplasm with a diffuse and granular appearance. BSC-1 cell monolayers were infected with the vaccinia virus vNSP1 or vT7lacOI at a multiplicity of infection (MOI) of 0.1 and induced with 1 mM isopropyl-beta-d-thiogalactopyranoside. At 15 h post-infection (hpi), the cells were processed using immunofluorescence (IF) with anti-NSP1 (white) and 4',6-diamino-2-phenylindole (DAPI) (blue). NSP1 was not detected in the cells infected with the parental vaccinia vT7lacOI (left picture), while in cells infected with vNSP1, a diffuse and granular appearance of NSP1 was observed (right picture); B: NSP1 localises to the cytoplasm in RRV-infected cells, with preferential localisation to the perinuclear region and granular appearance. MA-104 cell monolayers were infected with RRV at an MOI of 0.1 (upper row) or 1 (lower row). At 12 hpi, the cells were processed using IF with anti-NSP1 (white) and DAPI (blue). NSP1 localised preferentially to the perinuclear region, with some cells showing predominantly small granules (upper row, left picture) and others large granules (upper row, right picture). The extent of localisation differences and the heterogeneity in the levels of NSP1 expression in different cells are shown using a higher MOI (lower picture). Bar = 10 µ.

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Fig. 2
large non-structural protein 1 (NSP1) granules are detected only late in infection in rhesus rotavirus (RRV)-infected cells. MA-104 cells were mock-infected (row A) or infected with RRV at a multiplicity of infection of 0.1 (rows B-F) and at different times post-infection (pi) (as indicated), the cells were processed using immunofluorescence with anti-NSP1 (white) or anti-NSP2 (green) and 4',6-diamino-2-phenylindole (blue). Two images are shown for each pi time, which illustrate the heterogeneity in the intensity of NSP1 fluorescence. hpi: hours pi. Bar = 10 µ.

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Fig. 3
non-structural protein 1 (NSP1) has a low expression level that increases late in infection in rhesus rotavirus (RRV)-infected cells. MA-104 cells were mock-infected or infected with RRV at a multiplicity of infection of 10 and the cells were processed using Western blot analysis with anti-NSP1, anti-NSP2 and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at different times post-infection (pi) (as indicated). The anti-NSP1 serum detected an intense cellular band in mock-infected cells (Lane 1, panel A), whereas a specific NSP1 band was detected only in RRV-infected cells (A). The results of one representative WB assay are shown (B), with two lengths of film exposure [low (NSP1 L-EXP) or high (NSP1 H-EXP)], as well as quantitative analyses of three independent experiments normalising the band intensities of NSP1 to the loading control GAPDH (C). Standard deviations are shown above each bar. hpi: hours pi.

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Fig. 4
high levels of non-structural protein 1 (NSP1) expression in rhesus rotavirus (RRV)-infected cells correlate with altered viroplasm morphology. MA-104 cell monolayers were infected with RRV at a multiplicity of infection of 0.1. At 12 h post-infection (hpi), the cells were processed using immunofluorescence with anti-NSP1 (red) and one of three antibodies against proteins that localise in viroplasms (green): anti-viral protein (VP)6, anti-NSP2 or anti-VP2. The nuclei were visualised with 4',6-diamino-2-phenylindole (blue) (A). Representative images of normal or altered viroplasm morphology are shown, as indicated above the images. The association between high levels of NSP1 expression and altered viroplasm morphology was assessed at each pi time by classifying 150 rotavirus infected cells according to the level of NSP1 expression (low or high) and the distribution of NSP2 (normal or altered) (B). Bar = 10 µ.

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Fig. 5
high levels of non-structural proteins 1 (NSP1) expression in rhesus rotavirus (RRV)-infected cells depend on multiplicity of infection (MOI). MA-104 cells were infected with RRV at MOIs of 0.5, 2 or 8. To synchronise the infection, the cells were washed 1 h post-infection (hpi) and treated with the neutralising Mab 4F8 for 1 h at 4ºC, as previously described, to neutralise the input virus (Ruggeri & Greenberg 1991). At 15 hpi, the cells were processed using immunofluorescence with anti-NSP1 and the NSP1-expressing cells were classified according to the NSP1 expression level (low or high). The ratio of cells with high levels of NSP1 expression relative to all cells expressing NSP1 is shown. Standard deviations are shown for each MOI.

Fig. 1 intracellular localisation of rhesus rotavirus (RRV) non-structural protein 1 (NSP1). A: recombinant NSP1 localises to the cytoplasm with a diffuse and granular appearance. BSC-1 cell monolayers were infected with the vaccinia virus vNSP1 or vT7lacOI at a multiplicity of infection (MOI) of 0.1 and induced with 1 mM isopropyl-beta-d-thiogalactopyranoside. At 15 h post-infection (hpi), the cells were processed using immunofluorescence (IF) with anti-NSP1 (white) and 4',6-diamino-2-phenylindole (DAPI) (blue). NSP1 was not detected in the cells infected with the parental vaccinia vT7lacOI (left picture), while in cells infected with vNSP1, a diffuse and granular appearance of NSP1 was observed (right picture); B: NSP1 localises to the cytoplasm in RRV-infected cells, with preferential localisation to the perinuclear region and granular appearance. MA-104 cell monolayers were infected with RRV at an MOI of 0.1 (upper row) or 1 (lower row). At 12 hpi, the cells were processed using IF with anti-NSP1 (white) and DAPI (blue). NSP1 localised preferentially to the perinuclear region, with some cells showing predominantly small granules (upper row, left picture) and others large granules (upper row, right picture). The extent of localisation differences and the heterogeneity in the levels of NSP1 expression in different cells are shown using a higher MOI (lower picture). Bar = 10 µ.

Fig. 2 large non-structural protein 1 (NSP1) granules are detected only late in infection in rhesus rotavirus (RRV)-infected cells. MA-104 cells were mock-infected (row A) or infected with RRV at a multiplicity of infection of 0.1 (rows B-F) and at different times post-infection (pi) (as indicated), the cells were processed using immunofluorescence with anti-NSP1 (white) or anti-NSP2 (green) and 4',6-diamino-2-phenylindole (blue). Two images are shown for each pi time, which illustrate the heterogeneity in the intensity of NSP1 fluorescence. hpi: hours pi. Bar = 10 µ.

Fig. 3 non-structural protein 1 (NSP1) has a low expression level that increases late in infection in rhesus rotavirus (RRV)-infected cells. MA-104 cells were mock-infected or infected with RRV at a multiplicity of infection of 10 and the cells were processed using Western blot analysis with anti-NSP1, anti-NSP2 and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at different times post-infection (pi) (as indicated). The anti-NSP1 serum detected an intense cellular band in mock-infected cells (Lane 1, panel A), whereas a specific NSP1 band was detected only in RRV-infected cells (A). The results of one representative WB assay are shown (B), with two lengths of film exposure [low (NSP1 L-EXP) or high (NSP1 H-EXP)], as well as quantitative analyses of three independent experiments normalising the band intensities of NSP1 to the loading control GAPDH (C). Standard deviations are shown above each bar. hpi: hours pi.

Fig. 4 high levels of non-structural protein 1 (NSP1) expression in rhesus rotavirus (RRV)-infected cells correlate with altered viroplasm morphology. MA-104 cell monolayers were infected with RRV at a multiplicity of infection of 0.1. At 12 h post-infection (hpi), the cells were processed using immunofluorescence with anti-NSP1 (red) and one of three antibodies against proteins that localise in viroplasms (green): anti-viral protein (VP)6, anti-NSP2 or anti-VP2. The nuclei were visualised with 4',6-diamino-2-phenylindole (blue) (A). Representative images of normal or altered viroplasm morphology are shown, as indicated above the images. The association between high levels of NSP1 expression and altered viroplasm morphology was assessed at each pi time by classifying 150 rotavirus infected cells according to the level of NSP1 expression (low or high) and the distribution of NSP2 (normal or altered) (B). Bar = 10 µ.

Fig. 5 high levels of non-structural proteins 1 (NSP1) expression in rhesus rotavirus (RRV)-infected cells depend on multiplicity of infection (MOI). MA-104 cells were infected with RRV at MOIs of 0.5, 2 or 8. To synchronise the infection, the cells were washed 1 h post-infection (hpi) and treated with the neutralising Mab 4F8 for 1 h at 4ºC, as previously described, to neutralise the input virus (Ruggeri & Greenberg 1991). At 15 hpi, the cells were processed using immunofluorescence with anti-NSP1 and the NSP1-expressing cells were classified according to the NSP1 expression level (low or high). The ratio of cells with high levels of NSP1 expression relative to all cells expressing NSP1 is shown. Standard deviations are shown for each MOI.

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

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[1] + Corresponding author: lpadilla@unam.mx