However, the near normal export of the NP and PB2 mRNAs seen after NXF1 depletion or DRB treatment when NS1 expression is much reduced, as well as a consideration of the kinetics of protein expression in normal cells suggests that if this mechanism does operate at all, it does not do so for the intronless early gene transcripts
However, the near normal export of the NP and PB2 mRNAs seen after NXF1 depletion or DRB treatment when NS1 expression is much reduced, as well as a consideration of the kinetics of protein expression in normal cells suggests that if this mechanism does operate at all, it does not do so for the intronless early gene transcripts. METHODS Materials. 293T and MDCK cells were cultured as described previously (Hutchinson em et al. /em , 2008). for the localization of positive-sense RNA from segments 1, 4, 5, 7 and 8. Only background levels of transmission were seen from uninfected cells (Fig.?1a). In untreated cells all of the viral mRNAs tested were found to be predominantly cytoplasmic (Fig.?1b). When infected cells were treated with DRB, segment 5 mRNA still remained mostly cytoplasmic, while as expected (Amorim (2008) who found that NXF1 depletion of HEK cells did not MLN120B dramatically impact cell viability over the time-spans used here. The differing susceptibilities individual viral mRNAs showed to siRNA depletion of cellular export factors or DRB correlated better with the kinetic class of the viral gene product than with mRNA structure. Intronless transcripts for early gene products (in particular segment 5/NP mRNA) but also segment 1 (PB2) showed the least dependence on the NXF1 pathway (Fig.?7a), while late genes, including the intronless mRNA encoding HA, the spliced mRNA for M2 and the intron-containing but unspliced M1 message showing the clearest dependence (Fig.?7bCd). We have not examined the susceptibility of segment 6 (NA) mRNA to NXF1 depletion but Wang (2008) showed an association between the two molecules, while Hao (2008) reported that NXF1 depletion blocked expression of an artificial reporter mRNA based on segment 6. It therefore seems plausible that this NA mRNA has a comparable export mechanism to the HA mRNA (Fig.?7b). The correlation between the degree of dependence on MLN120B NXF1 and the kinetic class of the viral gene product is not perfect however, as expression of the late protein NS2 (from your spliced segment 8 mRNA) was less sensitive to DRB than expression MLN120B of the early protein NS1 from your unspliced transcript (Fig.?3b) and the export of the majority populace of positive-sense mRNA from segment 8 was inhibited by both DRB and NXF1 depletion (Figs?1, ?,44 and ?and55). The question therefore occurs of how the viral mRNAs are recruited to the NXF1/p15 pathway for export. Depletion of Aly, the most thoroughly characterized adaptor protein for cellular mRNA, experienced little effect on transport of viral messages (Figs?4 and ?and5)5) or protein expression (Fig.?3). This is perhaps surprising given the dependence cellular mRNAs show on Aly for export (Carmody & Wente, 2009; Cheng oocytes (Meignin & Davis, 2008), so we speculate that this reduction in HA expression seen here results from an effect downstream of mRNA nuclear export. Although we have shown that NXF1 and/or UAP56 are required for export of certain viral transcripts, MLN120B the mechanism(s) by which these factors are recruited to the mRNAs remains to be determined. Maturation of M2 mRNA resembles that of a Rabbit Polyclonal to ARNT normal cellular pre-mRNA: intron removal presumably leads to deposition of the exon junction complex, including UAP56, which will then recruit Aly and NXF1 (Fig.?7d). Alternatively or in addition, NXF1 might be directly recruited to the serine/arginine-rich protein splicing factor 2/alternative splicing factor (SF2/ASF) (Huang yet. Based on numerous precedents from other nuclear-transcribing viruses (Schneider & Wolff, 2009) it is also possible that viral polypeptide(s) act as an adaptor between the viral mRNA and the cellular nuclear export pathway. For instance, it has been suggested that the viral polymerase complex might functionally replace the cellular CBC for the purposes of nuclear export (Shih & Krug, 1996b). It is well established that the viral polymerase interacts with Pol II (Engelhardt em et al. /em , 2005; Loucaides em et al. /em , 2009; Mayer em et al. /em , 2007; Rameix-Welti em et al. /em , 2009), potentially placing it in the correct local environment to interact with the export machinery that would normally be MLN120B recruited co-transcriptionally to a cellular pre-mRNA. Such a mechanism is compatible with the observation that drugs that inhibit Pol II transcription inhibit export of most of the viral mRNAs (Amorim em et al. /em , 2007; Vogel em et al. /em , 1994; Wang em et al. /em , 2008; this study). NP is also a plausible adaptor candidate: non-RNP-associated NP shuttles between nucleus and cytoplasm (Elton em et al. /em , 2001; Neumann em et al. /em , 1997; Whittaker em et al. /em , 1996) as well as interacting with several cellular proteins involved in mRNA biogenesis and trafficking (Josset em et al. /em , 2008; Mayer em et al. /em , 2007; Momose em et al. /em , 2001). While our data here do not support a functionally.