Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses

Zina M Uckeley,Rebecca Moeller,Lars I Kühn,Emma Nilsson,Claudia Robens,Lisa Lasswitz,Richard Lindqvist,Annasara Lenman,Vania Passos,Yannik Voss,Christian Sommerauer,Martin Kampmann,Christine Goffinet,Felix Meissner,Anna K Överby,Pierre-Yves Lozach,Gisa Gerold

doi:10.1074/mcp.ra119.001631

Abstract

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

Highlights

Enveloped viruses are surrounded by a host cell derived lipid bilayer into which the viral glycoproteins are embedded [1]
Quantitative proteomics identifies GBF1 as UUKV glycoprotein interaction partner We have previously characterized UUKV G N/GC glycoprotein interactions with the C-type lectins DC-SIGN and L-SIGN, which are critical for virus entry [34,49]
In order to allow interactions of G N/GC with cell surface attachment factors, and with intracellular proteins contained in the lysate, we carried out the affinity purification of G N/GC and associating host proteins for 16 h in the cold

Summary

Introduction

Enveloped viruses are surrounded by a host cell derived lipid bilayer into which the viral glycoproteins are embedded [1]. To identify proteins which bind non- to the antibody matrix, we performed affinity purifications from cells that had been incubated with virus-free cell culture supernatants and used the same experimental conditions as for the UUKV containing samples.

Results

Conclusion