PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes

Abstract

It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 59UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1-eIF4GI and PABP1-eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs. INTRODUCTIONInfluenza virus infection efficiently shuts off the expression of the host cell genes (Skehel, 1972), while maintaining an efficient translation of viral proteins. During influenza virus infection, the virus evades the inhibition of protein synthesis through the inhibition of the double-stranded RNAactivated kinase (Lee et al., 1992;Lu et al., 1995;Polyak et al., 1996). Cellular protein synthesis shutoff may be the result of several alterations induced by the virus during infection. These include: (i) cap-snatching of cellular premRNAs (Krug et al., 1979), which probably contributes towards decreasing the synthesis of cellular mRNAs; (ii) inhibition of cleavage and polyadenylation of cellular premRNAs (Chen & Krug, 1999;Nemeroff et al., 1998); (iii) nuclear retention of poly(A)-containing cellular mRNAs (Fortes et al., 1994); (iv) degradation of cytoplasmic cellular mRNAs (Beloso et al., 1992;Inglis, 1982; Zürcher et al., 2000); and (v) preferential utilization of the translation machinery by the viral-specific mRNAs (Katze et al., 1986).Influenza virus mRNAs have a capped 59 end followed by a 10-12 nt long untranslated region of cellular, heterogeneous sequences generated by cap-snatching, which precede a viral-encoded, highly conserved sequence that is common to all influenza virus genes. The 39 end of the viral mRNAs is polyadenylated by a reiterative copy of a U 5-7 track present near the 59 end of the viral RNA (Luo et al., 1991;Poon et al., 1998Poon et al., , 1999Robertson et al., 1981). Although viral mRNAs are formally equivalent to cellular ones, influenza virus infection specifically enhances viral mRNA translation, with the conserved sequences contained within the 59-untranslated region (59UTR...

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

“…However, the NA reduction cannot be fully explained by the diminished transcription, and a reduction in mRNA translation has to be assumed to account for the reduction in NA accumulation. This would be in line with the proposed role for NS1 protein in the translation of late virus mRNAs (1,5,8,13,14,17). However, the results obtained with the 11C mutant indicate that there is a sequence-specific regulation of this NS1 function, as NA expression is affected but HA is not (Fig.…”

“…These include the virus RNP and/or polymerase (45), cellular proteins involved in translation such as hStaufen, PABPI, and eIF4G (1,5,16), and cellular factors involved in posttranscriptional processing of RNA such as CPSF (47) and NS1-BP, a potential splicing-related factor (72). These interactions may be responsible for the alterations in the control of cellular gene expression observed upon expression of NS1 cDNA (18,19,47,57), as well as for the regulation of virus gene expression (8,13).…”

Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

“…Interestingly, the wild-type NS1 protein of Sw/TX/98 virus has only 219 amino acids and lacks the poly(A)-binding protein binding domain that has been shown to be involved in inhibition of host cell mRNA polyadenylation (26). In addition, the NS1 proteins from the 1-126 and 1-99 viruses, but not the 1-73 virus, contain a domain that has been shown to bind to eukaryotic initiation factor 4GI and stimulate translation of viral mRNAs (1,4). Another function that has recently been shown is that the (50) needs to be further studied.…”

Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

“…This blocks the expression of host inhibitors, including interferon and tumor necrosis factor alpha (TNF-␣) (which were reduced in PR8-infected A549 cells) (Table 3), and thus a balance of host inhibition must be achieved while maintaining host gene transcription of mRNA and protein products employed for replication. Influenza NS1 protein also binds eIF4G1 and PABP1 translation initiation factors to favor influenza protein translation (9,17,69) relative to host translation. It is possible that the reduction in eIF4G1 as well as many ribosomal protein components may be involved in the mechanisms for preferential viral gene expression at the expense of host gene expression.…”