Influenza B and C virus NEP (NS2) proteins possess nuclear export activities

Abstract

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.

FIG. 1

Yeast two-hybrid analysis of NEP interactions. Influenza A, B, and C virus NEP proteins were fused to a LexA binding domain and used as bait to test for interaction with nucleoporins and Crm1, which were fused to an acidic activation domain (for details see Materials and Methods). A β-galactosidase gene containing upstream LexA binding sites was used as a reporter. The number of plus signs indicates the relative strength of blue color on 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside indicator plates. The scoring system is 1 for the weakest relative strength to 3 for the strongest relative strength. Rev (wild-type) and influenza A virus NEP protein results are included for comparison (6, 14, 37, 40, 44, 51)

FIG. 2

Influenza virus Rev*-NEP fusions can promote the nuclear export of CAT mRNA. (A) Full-length HIV-1 Rev (aa 1 to 116) contains both an RRE and a nuclear export signal (aa 75 to 83) and is therefore able to promote the nuclear export of pDM128 reporter mRNA. In Rev* (aa 1 to 69), the NES has been removed (44). Rev*, therefore, cannot interact with the cellular export machinery to promote the export of unspliced CAT mRNA containing an RRE. Fusion of the influenza A, B, and C virus NEP proteins (not drawn to scale) to Rev* results in the nuclear export of pDM128 mRNA, indicating the presence of a functional NES within the NEP. Rev*-ANEP contains 190 aa. Rev*-BNEP contains 191 aa. Rev*-CNEP contains 252 aa. (B) Thirty-five-millimeter-diameter dishes of 293T cells were transfected with pDM128 reporter (2 μg) and the indicated Rev* fusion plasmid (3 μg). Cell lysates were harvested 48 h posttransfection, and CAT assays were performed as described in Materials and Methods. CAT values are the mean percentages of duplicate transfections and are normalized to levels induced by Rev*-ANEP.

FIG. 3

Mapping of the NES for the influenza B virus NEP protein. (A) The amino acid sequence of influenza A and B virus NEP proteins are compared, and a 10-aa stretch with homology to the influenza A virus NEP NES was observed. Key hydrophobic residues (underlined) within the influenza B virus NEP NES motif were mutated to alanine and tested in the pDM128 CAT reporter assay. (B) CAT values are the mean percentages of duplicate transfections and are normalized to levels induced by Rev*-BNEP.

FIG. 4

Mapping of the influenza C virus NEP putative NES. (A) Within the C-terminal 94 aa, two Rev-like nuclear export signals were detected. Key hydrophobic amino acids (underlined) were mutated to alanine in each motif separately and tested in the CAT export assay. (B) A series of N- and C-terminal deletion mutants in the influenza C virus NEP ORF were made and fused to Rev*. The C-terminal 94 aa were found to be sufficient to promote the export of the CAT reporter gene. Plus signs indicate a positive CAT signal comparable to that of the full-length Rev*-CNEP fusion. A minus sign indicates CAT activity at or below background levels. (C) CAT values are the mean percentages of duplicate transfections and are normalized to levels induced by Rev*-CNEP.

FIG. 5

Effect of the NEP protein on the ability to package and passage a functional viral RNA-like CAT gene. 293T cells were transfected with either wild-type (WT) or NEP-lacking (−NEP) combinations of plasmids (Table 1). Forty-eight hours posttransfection clarified supernatants were used to infect MDCK cells. Cells were either superinfected with influenza B/Yamagata/73 virus at a multiplicity of 1 or mock infected with PBS. Twelve hours postinfection cells were harvested and assayed for CAT activity. The WT lane shows a positive signal for passage of the viral RNA-like CAT gene. The supernatant derived from the −NEP-transfected cells are not able to passage the viral RNA-like CAT gene. (A) CAT activity (10³ dilution of cell extract) from the primary transfection; (B) CAT activity (undiluted cell extract) detected following infection with clarified supernatants shown in panel A that were superinfected with influenza B/Yamagata/73 virus; (C) CAT activity (undiluted cell extract) from MDCK cells infected with clarified supernatants shown in panel A or mock superinfected with PBS.

FIG. 6

The NES contained in the influenza B virus NEP can be functionally replaced by the influenza A virus NEP NES. (A) Diagram of alterations of the influenza B virus NEP gene. B/delNES/NEP is the wild-type NEP with the first 20 aa removed. A MET codon has been added for proper protein translational initiation. B/NEP is the wild-type NEP gene from influenza B/Lee/40 virus. A21/B/NEP has the first 20 aa of the influenza B virus NEP replaced with the established NES contained within the first 21 aa of the influenza A virus NEP. Both ORFs were cloned into the vector pCAGGS. (B through D) 293T cells were transfected with each of the DNA combinations listed in Table 1. Forty-eight hours posttransfection supernatants were mixed with either 10⁶ PFU of influenza B/Yamagata virus or an equivalent volume of PBS and then used to infect fresh MDCK cells. (B) Primary transfection refers to the CAT activity (10³ dilution of cell extract) from the transfection of 293T cells. (C) Passage+Virus refers to the CAT activity (undiluted cell extract) detected following infection of MDCK cells with supernatants from the 293T transfection mixed with influenza B/Yamagata/73 virus. (D) Passage+PBS is the CAT activity from MDCK cells infected with supernatants from 293T transfected cells and mixed with PBS.