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. 2016 Dec;27(4):357-368.
doi: 10.1007/s13337-016-0347-3. Epub 2016 Sep 14.

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

Vibha Yadav  1 , Antonito T Panganiban  1 , Kerstin Honer Zu Bentrup  2 , Thomas G Voss  2
Affiliations

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

Vibha Yadav et al. Virusdisease. 2016 Dec.

Abstract

Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus replication.

Keywords: Golgi fragmentation; Influenza virus; Vesicular components.

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Figures

Fig. 1
Fig. 1
Golgi fragmentation after Influenza infection and redistribution of GM130 upon treatment with BFA, NOC, ZWEHD. A549 cells were pretreated with mock, NOC, ZWEHD and BFA for 1.5 h and then infected with Influenza at MOI 1.0. Slides were fixed with PFA and processed for indirect immunofluorecsence. a (i–iv) Cells were either stained with GM130 antibody alone to detect Golgi (red) or b (i–viii) costained with Influenza NP antibody (green) to detect intracellular Influenza infection. c (i–iv) Cells were either stained with Beta-Cop antibody alone to detect COPI (red) or d (i–viii) co-stained Influenza NP antibody (green) to detect intracellular Influenza infection
Fig. 2
Fig. 2
Live cell microscopy of Influenza infected cells. A549 cells were seeded on cover slips and stained for BODIPY-TR ceramide to detect Golgi and kept under microscope for focusing the cells and then NS1-GFP virus was provided to cells and made movie for 18 h
Fig. 3
Fig. 3
Influenza infection is inhibited in ldl-F cells at the non-permissive temperature. a CHO wild type and ldlF cells rescued with epsilon-COP were incubated at 34 °C or the non permissive temperature (40 °C) for 3 h. Cells were then left uninfected or infected with Influenza at MOI 1.0 for 4 h. Cells were fixed and processed for indirect immunofluorescence and cells were analyzed for Golgi makers, GM130 or β-COP (red) and NP Influenza protein (red). b ldlF cells were transfected with Rab1b-GFP and VSVG-GFP plasmid and kept the cells at the permissive temperature and infected the cells with Influenza at MOI 1.0 and fixed and processed for indirect immunofluorescence
Fig. 4
Fig. 4
Graph showing viral titer in supernatant of different inhibitors treated cells. A549 cells were treated with LB, DRB, combination of LB and DRB, NOC, ZWEHD, BFA for 30 min before infection and 1 h with or without Influenza infection and afterwards medium was changed and infection was proceed for next 24 h. Supernatants were collected and used for infecting MDCK cells. After 24 h, MDCK cells were fixed and processed for Fluorescent focusing assay
Fig. 5
Fig. 5
Colocalization of vesicular components during Influenza infection. a A549 cells were transfected with Rab1b-GFP, ARF1-RFP and GBF1-GFP plasmids. The cells were fixed and mounted and analyzed for GFP or RFP fluorescence. b Rab1b-GFP, ARF1-RFP and GBF1-GFP transfected cells were infected with influenza virus at an MOI of 1.0 for 24. Cells were fixed and analyzed for GFP or RFP fluorescence or the presence of NP Influenza protein
Fig. 6
Fig. 6
Effect of DRB, LB and combinatorial treatment on Golgi and NP distribution in Influenza infected cells. A549 cells were pretreated with LB, DRB and in combination for 30 min and cells were infected with Influenza MOI 1.0. Cells were fixed after 24 h p.i. with PFA and processed for indirect immunofluorescence. Golgi structure was stained with a murine monoclonal antibody against GM130 (red) and mouse monoclonal NP antibody was used to detect intracellular Influenza NP distribution (green)
Fig. 7
Fig. 7
A549 cells were transfected with VSVG-ts045-GFP-expressing plasmid and incubated at 40 °C overnight to accumulate VSVG-ts045-GFP protein in the ER. a Uninfected cells were either maintained at 40 °C or shifted to 32 °C for 15 or 30 min as indicated. The cells were subsequently fixed and mounted for fluorescence microscopy. b VSVG-ts045-GFP transfected cells were infected with influenza virus at an MOI 1.0 at 40 °C for 4 h and then shifted to 32 °C for 15 and 30 min. Cells were fixed and analyzed for GFP fluorescence or the presence of NP Influenza protein by indirect immunofluorecsence
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