The C-Terminal Domains of the PB2 Subunit of the Influenza A Virus RNA Polymerase Directly Interact with Cellular GTPase Rab11a

Abstract

Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this study, we utilized a combination of biochemical, proteomic, and biophysical approaches to characterize the interaction between the viral polymerase and Rab11a. Using pulldown assays, we showed that vRNPs but not complementary RNPs (cRNPs) from infected cell lysates bind to Rab11a. We also showed that the viral polymerase directly interacts with Rab11a and that the C-terminal two-thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627, and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, which has been shown to be important for binding Rab11 family-interacting proteins (Rab11-FIPs), is also important for PB2-C binding, suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs. IMPORTANCE The influenza virus RNA genome segments are replicated in the cell nucleus and are assembled into viral ribonucleoprotein (vRNP) complexes with viral RNA polymerase and nucleoprotein (NP). Replicated vRNPs need to be exported from the nucleus and trafficked across the cytoplasm to the cell membrane, where virion assembly takes place. The host GTPase Rab11a plays a role in vRNP trafficking. In this study, we showed that the viral polymerase directly interacts with Rab11a mediating the interaction between vRNPs and Rab11a. We mapped this interaction to the C-terminal domains of the PB2 polymerase subunit and the switch I region of Rab11a. Identifying the exact site of Rab11a binding on the viral polymerase could uncover a novel target site for the development of an influenza antiviral drug.

Keywords: PB2 polymerase subunit; RNA polymerase; Rab11a; cytoplasmic trafficking; influenza virus.

FIG 1

Recombinant Rab11a binds vRNPs from infected cell lysates. (A) Recombinant GST-Rab11a was analyzed by SDS-PAGE and proteins were visualized by staining with Coomassie brilliant blue. (B) HEK 293T cells were infected with influenza A/WSN/1933 (H1N1) (WSN) virus at a multiplicity of infection (MOI) of 5 or were mock infected, and whole-cell lysates were isolated at 12 h postinfection. Lysates were incubated with immobilized GST-Rab11a or GST. Viral RNAs isolated from cell lysates (input) and material bound to GST-Rab11a (bound) were analyzed by primer extension. 5S rRNA was used as a control. Note that 10-fold less cell lysate was used to prepare the input sample than for bound samples. (C) Quantitative analysis of viral RNAs bound to GST-Rab11a or GST. Data are means ± standard deviations (n = 3 independent experiments). (D) Mass spectrometry-based proteomic analysis and label-free relative quantification of viral proteins bound to Rab11a or GST. A two-tailed t test was used to calculate P values (n = 2 independent experiments).

FIG 2

The RNA polymerase of IAV directly interacts with Rab11a. Purified recombinant polymerase from A/Northern Territory/60/1968 (H3N2) (NT60) (A), A/Brevig Mission/1/1918 (H1N1) (BM18) (D), and A/duck/Fujian/01/2002 (H5N1) (Fj02) (G). GST-Rab11a (B, E, and H), and GST tag (C, F, and I) were immobilized on glutathione Sepharose beads before being incubated with a purified NT60 (B and C), BM18 (E and F) or Fj02 (H and I) polymerase. Proteins were released using GST-tagged 3C protease (GST-3C) to cleave off Rab11a from the GST tag. Purified polymerase (purified), unbound flowthrough polymerase (FT), proteins bound to GST-Rab11a or GST tag (bound), proteins released upon cleavage with GST-tagged 3C protease (GST-3C) (cleaved), and proteins remaining associated with the beads after cleavage (beads) were analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie brilliant blue.

FIG 3

A complete C-terminal region of the PB2 polymerase subunit is required for Rab11a binding. (A) Schematics of full-length PB2, PB2 lacking the 627 domain (PB2_Δ535–667), and PB2 fragments 627-NLS and PB2-C. (B to D) The 627 domain of PB2 is required for interaction between the influenza virus polymerase and Rab11a. (E to G) The PB2 627-NLS domain alone does not bind Rab11a. (H to J) The C-terminal domains of PB2 (PB2-C) mediate interaction with Rab11a. (B, E, and H) Purified recombinant polymerase from A/Northern Territory/60/1968 (H3N2) (NT60) lacking the 627 domain (PB2_Δ535–667) (B) and purified recombinant 627-NLS (E) and PB2-C (H) fragments of NT60 PB2. GST-Rab11a (C, F, and I) or GST tag (D, G, and J) were immobilized onto glutathione Sepharose beads before being incubated with a purified PB2_Δ535–667 (C and D), 627-NLS (F and G), or PB2-C (I and J). Proteins were released by using GST-tagged 3C protease (GST-3C) to cleave off Rab11a from the GST tag. Purified polymerase or polymerase fragments (purified), unbound flowthrough polymerase or fragments (FT), proteins bound to GST-Rab11a or GST tag (bound), proteins released upon cleavage with GST-tagged 3C protease (GST-3C) (cleaved), and proteins remaining associated with the beads after cleavage (beads) were analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie brilliant blue.

FIG 4

The switch I region of Rab11a is important for PB2 binding. (A) Crystal structure of Rab11a in complex with Rab11 family-interacting protein 2 (Rab11-FIP2) showing the involvement of the switch I region in the interaction. The three amino acids in the switch I region at the Rab11-FIP2 interaction interface are indicated. Mg²⁺ ions are shown as pink spheres. The PDB code is 2GZD. (B) GST-Rab11a_A44-46 mutant (top) and wild-type Rab11a (bottom) were immobilized on glutathione Sepharose beads and incubated with PB2-C derived from the polymerase of influenza A/Northern Territory/60/1968 (H3N2) (NT60) virus. Proteins were released by using GST-tagged 3C protease (GST-3C) to cleave off Rab11a from the GST tag. Unbound flowthrough PB2-C (FT), proteins bound to GST-Rab11a_A44-46 or Rab11a (bound), proteins released upon cleavage with GST-tagged 3C protease (GST-3C) (cleaved), and proteins remaining associated with the beads after cleavage (beads) were analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie brilliant blue.

FIG 5

PB2-C and Rab11a_1-173 form a 1:1 stoichiometric complex in solution. (A) The C-terminal disordered tail of Rab11a is not required for IAV polymerase binding. Recombinant truncated GST- Rab11a_1-173 lacking the C-terminal 43 amino acid residues was immobilized on glutathione Sepharose beads and incubated with polymerase of influenza A/Northern Territory/60/1968 (H3N2) (NT60) virus. Proteins were released by using GST-tagged 3C protease (GST-3C) to cleave off Rab11a from the GST tag. Unbound flowthrough polymerase (FT), proteins bound to GST-Rab11a_1-173 (bound), proteins released upon cleavage with GST-tagged 3C protease (GST-3C) (cleaved), and proteins remaining associated with the beads after cleavage (beads) were analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie brilliant blue. (B to E) SEC-SAXS analysis of Rab11a, PB2-C, and PB2-C–Rab11a_1-173 complex. Chromatograms from the differential refractive index detector are shown for PB2-C, Rab11a, and the PB2-C–Rab11a_1-173 complex at 1:0.5, 1:1, and 1:2 molar ratios. Blue and red vertical lines indicate PB2-C and Rab11a_1-173 elution peaks, respectively (B). Scattering profiles, Kratky plots, and pair distance distribution function of Rab11a_1-173, PB2-C, and the PB2-C–Rab11a_1-173 complex are normalized and shown in panels C to E, respectively.