Volume 1, Issue 7, Pages 991-1000 (June 1998) Influenza Virus NS1 Protein Interacts with the Cellular 30 kDa Subunit of CPSF and Inhibits 3′ End Formation of Cellular Pre-mRNAs  Martin E. Nemeroff, Silvia M.L. Barabino, Yongzhong Li, Walter Keller, Robert M. Krug  Molecular Cell  Volume 1, Issue 7, Pages 991-1000 (June 1998) DOI: 10.1016/S1097-2765(00)80099-4

Figure 1 Identification of a Cellular Protein that Binds Specifically to the Effector Domain of the Influenza Virus NS1 Protein (A) Yeast two-hybrid results. The indicated NS1 DNA in the Gal4 DNA-binding domain vector (the prey) was cotransformed into yeast with Neb1 cDNA in the Gal4 activation domain vector (the bait), and transformants were assayed for β-galactosidase activity in liquid cultures (Miller unitsMiller 1972). (B) The Neb1 protein binds directly to the effector domain of the viral NS1 protein in vitro. GST-Neb1 protein (1 μg) was incubated with 10 μl of 35S-labeled NS1 wild-type protein (lane 2), NS1ΔR (lane 4), or NS1ΔE (lane 6) in the presence of 20 μl of glutathione Sepharose 4B beads. The labeled proteins eluted from the beads were analyzed by electrophoresis on a 14% SDS–polyacrylamide gel: S, selected protein; U, unselected protein. Lanes 1, 3, and 5 each contain 2 μl of the indicated 35S-labeled NS1 protein. The NS1 proteins synthesized in vitro run as doublets, because a portion of these proteins is phosphorylated by the reticulocyte extracts. (C) The amino acid sequences of the Neb1 protein and the CPSF 30 kDa subunit are virtually identical. DNA sequencing of Neb-1 DNA was performed with the dideoxy chain termination method. The deduced amino acid sequence of the Neb-1 open reading frame was aligned with the amino acid sequence of the bovine CPSF 30 kDa subunit (Barabino et al. 1997). Amino acids shared between the two proteins are boxed. Zinc finger and zinc knuckle motifs are indicated by shading, and the cysteine (C) and histidine (H) residues in these motifs are highlighted. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 2 The Viral NS1 Protein in Infected Cells Is Physically Associated with 30 kDa Protein Molecules that Are in CPSF Complexes Containing All Four Protein Subunits (A) The NS1 protein and CPSF 30 kDa proteins are physically associated in infected cells. 35S-labeled total cell extracts from influenza virus–infected cells were immunoprecipitated with CPSF 30 kDa antiserum (lane 2), preimmune control serum (lane 3), or NS1 antiserum (lane 4), and the immunoprecipitates were analyzed by electrophoresis on an SDS–polyacrylamide gel. Lane 1: infected cell extract prior to immunoprecipitation. 35S-labeled NS1 protein synthesized in vitro (lane 5) was immunoprecipitated with CPSF 30 kDa antiserum (lane 6). Positions of the viral nucleocapsid (NP) protein and NS1 proteins are indicated. (B) Antiserum against the NS1 protein coprecipitates the other subunits of CPSF from infected cell nuclear extracts. Unlabeled nuclear extracts from influenza virus–infected cells were immunoprecipitated with NS1 antiserum (αNS1, lane 2) or preimmune control serum (αC, lane 3). The immunoprecipitates were subjected to gel electrophoresis. Lane 1, marker (M): aliquot of a partially purified fraction from a HeLa cell nuclear extract (Maldonado et al. 1996). The proteins in the gel were transferred to nitrocellulose, which was probed with antisera against the 73 kDa and 100 kDa CPSF subunits. Antibodies were stripped from the nitrocellulose, which was then probed with affinity-purified anti–160 kDa antibodies. Lane 4, marker (M); lane 5, anti-NS1 immunoprecipitate (αNS1); lane 6, control immunoprecipitate (αC). The major bands near the bottom of the gel are antibody heavy chains. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 3 The NS1 Protein Inhibits the Cleavage and Polyadenylation of Pre-mRNA In Vitro (A) Inhibition of cleavage of L3 pre-mRNA. HeLa cell nuclear extract was preincubated for 10 min at 30°C with increasing amounts of the NS1 protein (15, 30, 75, 150, 225, and 300 ng: lanes 2–7, respectively). The cleavage reaction was then carried out for 1 hr at 30°C, and the products were separated on a 6% denaturing polyacrylamide gel. Marker: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: full-length L3 pre-mRNA substrate. Position of the upstream (5′) cleavage product is indicated. (B) Inhibition of polyadenylation of precleaved L3 pre-mRNA. Purified CPSF (50 fmol) and poly(A) polymerase (280 fmol) were preincubated with increasing amounts of the NS1 protein (15, 30, 75, 150, 225, and 300 ng: lanes 2–7, respectively). Precleaved L3 pre-mRNA was added, and after a 30 min incubation at 30°C, the products were separated on a 12% denaturing polyacrylamide gel. Markers: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: precleaved RNA substrate. Positions of the polyadenylated mRNA products (pA) are indicated. (C) The effector domain of the NS1 protein is required for the inhibition of pre-mRNA polyadenylation in vitro. Polyadenylation reactions were preincubated with increasing amounts (15, 30, 75, and 150 ng) of the NS1RM protein (lanes 2–5), the NS1ΔE protein (lanes 6–9), or the NS1EM protein (lanes 10–13). Precleaved L3 pre-mRNA was added, and after a 30 min incubation at 30°C,the products were separated on a 12% denaturing polyacrylamide gel. Markers: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: precleaved RNA substrate. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 3 The NS1 Protein Inhibits the Cleavage and Polyadenylation of Pre-mRNA In Vitro (A) Inhibition of cleavage of L3 pre-mRNA. HeLa cell nuclear extract was preincubated for 10 min at 30°C with increasing amounts of the NS1 protein (15, 30, 75, 150, 225, and 300 ng: lanes 2–7, respectively). The cleavage reaction was then carried out for 1 hr at 30°C, and the products were separated on a 6% denaturing polyacrylamide gel. Marker: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: full-length L3 pre-mRNA substrate. Position of the upstream (5′) cleavage product is indicated. (B) Inhibition of polyadenylation of precleaved L3 pre-mRNA. Purified CPSF (50 fmol) and poly(A) polymerase (280 fmol) were preincubated with increasing amounts of the NS1 protein (15, 30, 75, 150, 225, and 300 ng: lanes 2–7, respectively). Precleaved L3 pre-mRNA was added, and after a 30 min incubation at 30°C, the products were separated on a 12% denaturing polyacrylamide gel. Markers: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: precleaved RNA substrate. Positions of the polyadenylated mRNA products (pA) are indicated. (C) The effector domain of the NS1 protein is required for the inhibition of pre-mRNA polyadenylation in vitro. Polyadenylation reactions were preincubated with increasing amounts (15, 30, 75, and 150 ng) of the NS1RM protein (lanes 2–5), the NS1ΔE protein (lanes 6–9), or the NS1EM protein (lanes 10–13). Precleaved L3 pre-mRNA was added, and after a 30 min incubation at 30°C,the products were separated on a 12% denaturing polyacrylamide gel. Markers: end-labeled HpaII-digested pBR322 fragments (M). Lane 1: precleaved RNA substrate. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 4 The NS1 Protein Prevents the CPSF Complex from Binding to the L3 Pre-mRNA Substrate CPSF (10 fmol) was preincubated for 10 min at 30°C with 25 or 75 ng of either wild-type NS1 protein (lanes 3 and 4), NS1RM protein (lanes 5 and 6), NS1ΔE protein (lanes 7 and 8), or the NS1EM protein (lanes 9 and 10). Each mixture was then incubated for 30 min at 30°C with 32P-labeled L3 pre-mRNA, and the reactions were analyzed by electrophoresis on a 3% acrylamide/0.5% agarose nondenaturing polyacrylamide gel. Lane 2: CPSF in the absence of the NS1 protein. Lane 1: labeled L3 pre-mRNA substrate alone. The position of the CPSF–L3 pre-mRNA complex is indicated. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 5 The NS1 Protein Inhibits 3′ End Cleavage of Pre-mRNAs In Vivo (A) Diagram of the uniformly labeled RNA probe, and the predicted sizes of the RNA fragments protected by the two uncleaved and two cleaved pre-mRNAs. (B) 293 cells were cotransfected with 5 μg of the pctat plasmid and with 15 μg of the indicated pBC12 plasmid: NS1dm (lane 1); NS1 (lane 2); or NS1ΔE (lane 3). At 40 hr posttransfection, the cells were collected, and total RNA was analyzed by ribonuclease protection. Lane 2*: longer exposure of lane 2. Sizes of the protected fragments were estimated based on the migration of DNA molecular weight markers. (C) 293 cells were cotransfected with the pctat plasmid and with the NS1dm (lanes 1 and 2) or NS1 (lanes 3 and 4) plasmid. At 40 hr posttransfection the cells were fractionated into nucleus (N) and cytoplasm (C). The RNA extracted from these two fractions was analyzed by ribonuclease protection. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 6 The NS1 Protein Inhibits 3′ Polyadenylation of Pre-mRNAs In Vivo (A) Diagram of the uniformly labeled RNA probe, and the predicted sizes of the RNA fragments protected by the polyadenylated pctat/NS1 mRNAs and the corresponding nonpolyadenylated pre-mRNAs. (B) 293 cells were cotransfected with 5 μg of the plasmid (pctat) encoding spliced tat mRNA and with 15 μg of the indicated pBC12 plasmid: NS1dm (lane 1); NS1 (lane 2); or NS1ΔE (lanes 3). At 40 hr posttransfection, the cells were collected and total RNA was analyzed by ribonuclease protection. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)

Figure 7 Proposed Mechanism of Selective Inhibition of the Nuclear Export of Cellular, and Not Viral, mRNAs Resulting from the Interaction of the Influenza Viral NS1 with the Cellular CPSF 30 kDa Protein Binding of the NS1 protein to the 30 kDa subunit of CPSF results in the inhibition of 3′ cleavage and polyadenylation (1) and of nuclear export (2) of host pre-mRNAs, which are cleaved by the virion cap-dependent endonuclease to produce the primers required for viral mRNA synthesis. The 3′-terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of 4–7 Us in the virion RNA templates. The poly(A)-containing viral mRNAs are exported from the nucleus. Molecular Cell 1998 1, 991-1000DOI: (10.1016/S1097-2765(00)80099-4)