Summary of viral replication kinetics, nuclear localization, nuclear export capacity, and CRM1 binding of the NP-NES3 consensus sequence mutants.

a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g004" target="_blank">Fig. 4C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>% cell count ± SD with nuclear localization of NP from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g005" target="_blank">Fig. 5B</a>.</p>d<p>−, ±, + indicate not occur, partially occur, occur of nuclear export capacity, respectively derived from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g006" target="_blank">Fig. 6</a>.</p>e<p>% intensity of the pull-downed NP band compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g007" target="_blank">Fig. 7C</a>.</p

Summary of viral replication kinetics and nuclear localization of NES consensus sequence mutants.

a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g001" target="_blank">Fig. 1C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>–, ±, + indicate no change, partial change, great change of NES mutant protein localization compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g002" target="_blank">Fig. 2</a>.</p

Construction and expression of NP-NES3 consensus sequence mutants.

<p>(A) NP-NES harboring mutations in individual or all Φ residues (leucine was replaced by alanine by site-directed mutagenesis). (B) Expression of wild-type NP and NP-NES3 mutants (Φ1, Φ2, Φ3, and Φ4) was compared by Western blotting with an anti-WSN Ab and an anti-actin MAb.</p

Nuclear export activity of NP-NES3 mutant proteins.

<p>HeLa cells growing on cover glass were transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h. The cells were then permeabilized with 50 µg/ml digitonin for 5 min on ice. The cytoplasmic components, including NP, were removed by washing (only NP in the nucleus remained). The nuclear export activity of NP was allowed to proceed in the presence of fresh total HeLa cell lysate at 30°C for 1 h (left column). Negative controls were incubated in the absence of total cell lysate (right panel). The cells were then stained with an anti-NP MAb followed by anti-mouse Alexa Flour 488 and Hoechst 333342, and observed under a confocal laser-scanning microscope. The white arrow head indicates nuclear export activity, whereas the yellow arrow head indicates a failure of nuclear export activity.</p

List of NES consensus sequence mutants.

a<p>Hydrophobic (Φ) residues, e.g., leucine, isoleucine, valine, and methionine, in the NES consensus sequence are underlined.</p

Intracellular localization of NP-NES3 mutant proteins.

<p>(A) HeLa cells were grown on cover glass and transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h before immunofluorescence staining with an anti-NP MAb followed by anti-mouse Alexa Fluor 488 and Hoechst 333342. The cells were then observed under a confocal laser-scanning microscope. The white and yellow arrow heads indicate predominant localization of NP in the cytoplasm (cytoplasmic staining > nuclear staining) and nucleus (nuclear staining > cytoplasmic staining), respectively. (B) Nuclear localization of NP wild-type and NP-NES3 mutants from A. Data are presented as the percentage (± SD) of total cell count with predominant nuclear or cytoplasmic staining of NP from five separate fields.</p

Conservation of hydrophobic (Φ) residues within the NP-NES3 consensus sequences of influenza A viruses.

<p>Conservation of hydrophobic (Φ) residues within the NP-NES3 consensus sequences of influenza A viruses.</p

Production, protein expression, and replication kinetics of NES mutant viruses.

<p>(A) Wild-type NES or mutant viruses (M1-NES, NS1-NES, NS2-NES1, NS2-NES2, NP-NES1, NP-NES2, and NP-NES3) were produced via reverse genetics by co-culturing HEK-293T and MDCK cells transfected with eight viral genomic plasmids (pHH21 containing PA, PB1, PB2, HA, NA, NP, M, and NS) and four viral protein expression plasmids (pCAGGS containing PA, PB1, PB2, and NP) for 72 h. Supernatants containing viruses were collected, clarified, and titrated in a plaque assay on MDCK cells. Viral titration was performed in triplicate, and the plaque forming units PFU/ml (mean±SD) was calculated and plotted (upper panel). Viral protein expression in the producer cells was compared by collecting the cells, lysing them, and separating the proteins on 10% SDS-PAGE gels. Proteins were then blotted with either an anti-WSN antibody (Ab) or an anti-actin monoclonal antibody (MAb) (lower panel). The HA, NP, M1, and M2 proteins are shown alongside molecular weight markers. (B) Expression of NP-NES wild-type and mutant proteins was compared by transfecting NP/pHH21, NP/pCAGGS, PA/pCAGGS, PB1/pCAGGS, or PB2/pCAGGS into HEK-293T cells for 48 h. The cells were then lysed and blotted with either an anti-WSN Ab or an anti-actin MAb. (C) MDCK cells were infected with equal amounts of each of the viruses (MOI = 0.001) described in (A) and then cultured. At the indicated time-points post-infection, the viruses were collected and titrated in a plaque assay. Data are expressed as the mean (±SD) PFU/ml from triplicate titrations.</p

Binding of NP-NES3 mutant proteins to CRM1.

<p>(A) FLAG-tagged NP-NES3 wild-type and mutant (Φ1, Φ2, Φ3, and Φ4) proteins were prepared by transfecting the relevant pCAGGS plasmids into HEK-293T cells. The cells were then lysed. The proteins were captured by anti-FLAG agarose beads and eluted with a FLAG-peptide. CRM1-HA agarose beads were prepared by transfecting CRM1-HA/pCAGGS into HEK-293T cells. The cells were then lysed and the proteins were captured on anti-HA agarose beads. The purified proteins and beads were then run in 10% SDS-PAGE gels and stained with Coomassie Brilliant Blue. (B) NP/CRM1 binding was demonstrated by incubating equal amounts of CRM1-HA agarose beads or anti-HA agarose beads alone with purified NP proteins at 4°C for 3 h. After pull-down and washing, the beads were boiled with 4×SDS sample buffer and subjected to 10% SDS-PAGE and Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. Input NP at 30% was included. (C) Equal amounts of purified wild-type NP-NES3-FLAG or mutant (Φ1, Φ2, Φ3, and Φ4) protein were co-incubated with CRM1-HA agarose beads at 4°C for 3 h, pulled-down, washed, boiled with 4×SDS sample buffer, and then subjected to Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. NP/CRM1 binding affinity was compared by measuring the intensity of the NP band normalized against the CRM1 band. Each 30% input NP protein and remained NP protein after binding with the CRM1-HA agarose beads are also shown.</p

HIV-1 Vpr Abrogates the Effect of TSG101 Overexpression to Support Virus Release

<div><p>HIV-1 budding requires interaction between Gag and cellular TSG101 to initiate viral particle assembly and release via the endosomal sorting complexes required for transport (ESCRT) pathway. However, some reports show that overexpression of TSG101 inhibits virus release by disruption of Gag targeting process. Since a HIV-1 accessory protein, Vpr binds to Gag p6 domain at the position close to the binding site for TSG101, whether Vpr implicates TSG101 overexpression effect has not been investigated. Here, we found that Vpr abrogates TSG101 overexpression effect to rescue viral production. Co-transfection of TSG101 and Gag with Vpr prevented TSG101-induced Gag accumulation in endosomes and lysosomes. In addition, Vpr rescued virus-like particle (VLP) production in a similar manner as a lysosomal inhibitor, Bafilomycin A1 indicating that Vpr inhibits TSG101-induced Gag downregulation via lysosomal pathway. Vpr and Gag interaction is required to counteract TSG101 overexpression effect since Vpr A30F mutant which is unable to interact with Gag and incorporate into virions, reduced ability to prevent Gag accumulation and to rescue VLP production. In addition, GST pull-down assays and Biacore analysis revealed that Vpr competed with TSG101 for Gag binding. These results indicate that Vpr overcomes the effects of TSG101 overexpression to support viral production by competing with TSG101 to bind Gag.</p></div