Export of Adenoviral Late mRNA from the Nucleus Requires the Nxf1/Tap Export Receptor

Abstract

One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infectedcell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.During the late phase of productive infection by human species C adenovirus, such as adenovirus type 5 (Ad5), viral late mRNAs are exported selectively from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNAs (see references 6, 28, and 34 for reviews). Such regulation of mRNA export requires the viral E1B 55-kDa protein (3, 67, 71) and the complex it forms with the E4 Orf 6 protein (13,23,87). In both transformed and normal human cells, efficient export of viral late mRNAs correlates with the E4 Orf6 protein-dependent recruitment of the E1B protein to the peripheral zones of viral replication centers (40,41,66), which are the sites of synthesis and at least initial processing of viral late pre-mRNAs (2,14,68,69). In infected cells, the E1B 55-kDa and E4 Orf6 proteins associate with the cellular proteins cullin 5, elongins B and C, and Rbx to form an E3 ubiquitin ligase (46). Substrates of the infected-cell-specific ubiquitin ligase include the cellular p53 protein (21,46,61,71), the Mre11, Rad50, and Nbs1 components of the MRN complex (85), DNA ligase IV (5), and integrins ␣3 (25), which are targeted for proteasomal degradation. The infected-cell-specific ligase has also been implicated in regulation of mRNA export during the late phase of infection. Synthesis in infected cells of a dominant-negative derivative of cullin 5 that stabilized p53 and Mre11 resulted in decreases in ...

Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

“…Our recent RNA chromatin immunoprecipitation assays suggest that agnoprotein is an RNA binding protein (data not shown) rather than a DNA binding protein (75). As mentioned above, we believe that dimeric and oligomeric forms of agnoprotein may play important roles in the transport of JCV transcripts from the nucleus to the cytoplasm in a Crm1-dependent (72,73) or -independent (76) manner. This possibility will be further investigated.…”

Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

“…Furthermore, its interaction with the HAdV-C5 E4 Orf6 protein has been reported to promote cytoplasmic accumulation of ARE-containing mRNAs via an XPO1-independent pathway (Higashino et al, 2005). The selective export of viral late mRNAs is also XPOl independent (Flint et al, 2005; Yatherajam et al, 2011) and requires the E4 Orf6 protein (Blanchette et al, 2008; Woo and Berk, 2007) raising the possibility that ANP32A might participate in regulation of mRNA export in HAdV-C5-infected cells. We therefore investigated the effect of RNAi-mediated inhibition of ANP32A synthesis on viral late gene expression, by monitoring the accumulation of the late protein, protein V (Berk, 2013), as it is well established that the impaired export of viral late mRNAs in cells infected by E1B 55 kDa-null mutant viruses leads to substantial defects in late protein synthesis (Babiss et al, 1985; Harada and Berk, 1999; Pilder et al, 1986; Williams et al, 1986).…”

“…Selective export of viral late mRNAs requires formation of the E1B 55 kDa protein-containing E3 ubiquitin ligase (Blanchette et al, 2008; Woo and Berk, 2007) and the major cellular mRNA export receptor NXF1 (Yatherajam et al, 2011), but the relevant substrate(s) of the virus-specific ligase have not been identified. The NXF1 receptor is not targeted for proteasomal degradation in HAdV-C5-infected cells, nor are two subunits (THOC1 and THOC4) of the TREX complex (Yatherajam et al, 2011), which couples export to prior reactions in mRNA biogenesis (Katahira, 2012; Reed and Cheng, 2005). One class of proteins enriched in the E1B 55 kDa protein-associated population is defined by the GO term RNA splicing (Table 2).…”

DNA Virus Replication Compartments

“…Since during Ad replication the transport of bulk cellular mRNA is simultaneously blocked (193,194), it is likely that one or more proteins implicated in their export are targeted for proteasomal degradation, which might, in turn, promote preferential export of viral transcripts. Second, further studies suggested that the cellular export receptor of bulk cellular mRNA, TAP/NXF1, participates in the export of Ad late transcripts (195). An interesting hypothesis is that cellular export factors are relocalized to viral transcription and replication centers and thus promote efficient export of newly transcribed mRNAs out of the nucleus.…”