Functional Analysis of GRSF1 in the Nuclear Export and Translation of Influenza A Virus mRNAs

Abstract

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.

Keywords: GRSF1; host adaptation; influenza A virus; mRNA nuclear export; translation.

Figure 1

Attenuated NPCA virus growth in cultured cells. (a) Schematic of GRSF1 binding site AGGGU and nucleotide identity in pH1N1 Cal WT gene segments. Sequences of 5’UTR of each viral segment are shown as positive sense. Nucleotides at 10–14 of each gene segment are underlined or bolded. (b) A single-step growth curve of indicated viruses in A549 cells infected at an MOI of 0.2. At indicated times, virus in the supernatant was collected and titrated. (c) MDCK cells were infected with the indicated viruses at an MOI of 0.01 and cultured in the presence of acetylated trypsin. At indicated times, virus in the supernatant was collected and titrated. All error bars show means plus/minus the standard deviations (n  =  3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparison test in PRISM (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 2

Viral protein expression in infected cells. (a) A549 cells were infected at an MOI of 2 and cell lysates were collected at 10 and 24 hpi. Representative image of immunoblot analysis of PB1, PA, NP, M1, and β-actin in cell lysates using specific antibodies. M: mock infection. (b–e) Relative viral protein expression was calculated by densitometry analyses, normalized to actin, and expressed as each respective Cal NPCA protein expression relative to Cal WT. All error bars show means plus/minus the standard deviations (n  =  5 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparison test in PRISM (*** p  <  0.001, **** p < 0.0001).

Figure 3

RNA production in infected cells. A549 cells were infected with indicated viruses at an MOI of 0.2 At indicated times, total RNA was extracted, and Cal NP mRNA (a), cRNA (b), and vRNA (c) were quantitated by strand-specific qRT-PCR. All error bars show means plus/minus the standard deviations (n  =  3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparison test in PRISM (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 4

Reduced nuclear export of NP mRNA in NPCA virus-infected cells. A549 cells were infected at an MOI of 3. At 8 hpi, cells were fractionated into nuclear and cytosolic fractions and RNAs were quantitated by qRT-PCR. (a) Percentages of GAPDH mRNA and U6 snRNA in cytosolic fraction. (b,c) NP (b) and PB1 (c) mRNAs in nuclear and cytosolic fractions were quantified and total mRNAs were calculated. (d–f) Quantities of NP mRNA (d), PB1 mRNA (e), and NP vRNA (f) in nuclear and cytosolic fractions. All error bars show means plus/minus the standard deviations (n  =  3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparison test in PRISM (* p  <  0.05).

Figure 5

Reduced ribosome association and translation of NPCA mRNA. (a) Initially, 293T cells were transfected with NP WT or NPCA mRNA transcribed in vitro. NP protein in transfected cells at 4 h post transfection was determined by Western blot analysis. Relative NP expression was calculated by densitometry analyses and normalized to actin. The data are expressed as NP expressed from NPCA mRNA relative to NP WT mRNA. All error bars show means plus/minus the standard deviations (n  =  3 biological replicates). Unpaired two–tailed t–test in PRISM (** p  <  0.01). (b) Representative polysome traces from 293T cells transfected with NP WT mRNA (black) or NPCA mRNA (red). (c–f) NP mRNA and cellular GAPDH mRNA in fractionated cell lysates were quantified by qRT-PCR and grouped into free (fractions 1–5), ribosome (fractions 6–9), light polysomes (fractions 10–12), medium polysomes (13–15), or heavy polysomes (fractions 16–18). (c) Total NP mRNA was calculated by summing all fractions. (d) NP mRNAs are presented as value per grouped fractions and percentage of total NP mRNA. (e) Total GAPDH mRNA was calculated by summing all fractions. (f) GAPDH mRNAs are presented as value per grouped fractions and percentage of total GAPDH mRNA (n  =  1 biological replicate).