Stefan Windhaber, Qilin Xin, Zina M Uckeley, Jana Koch, Martin Obr, Céline Garnier, Catherine Luengo-Guyonnot, Maëva Duboeuf, Florian K M Schur, Pierre-Yves Lozach
With more than 80 members worldwide, the genus in the family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.
J Cell Biol. 1980 Feb;84(2):404-20
[PMID:
6991511]
J Gen Virol. 1976 Jan;30(1):123-30
[PMID:
1245842]
Virology. 1995 Feb 20;207(1):136-42
[PMID:
7871722]
Viruses. 2021 May 10;13(5):
[PMID:
34068494]
J Virol. 2014 Nov;88(22):13029-46
[PMID:
25165113]
EMBO J. 2021 Aug 16;40(16):e107821
[PMID:
34159616]
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11876-81
[PMID:
16081529]
Traffic. 2016 Jun;17(6):639-56
[PMID:
26990254]
J Biol Chem. 2019 May 3;294(18):7335-7347
[PMID:
30804209]
Virology. 1996 Nov 1;225(1):243-7
[PMID:
8918555]
PLoS Pathog. 2013;9(5):e1003374
[PMID:
23696739]
Virology. 2005 Jul 20;338(1):121-32
[PMID:
15923017]
Antimicrob Agents Chemother. 2020 May 21;64(6):
[PMID:
32284379]
J Virol. 1987 Jul;61(7):2319-21
[PMID:
3586135]
Nat Commun. 2019 Feb 20;10(1):879
[PMID:
30787296]
Clin Infect Dis. 2021 May 18;72(10):1701-1708
[PMID:
32516409]
J Virol. 2020 Aug 17;94(17):
[PMID:
32522852]
Viruses. 2020 Apr 04;12(4):
[PMID:
32260371]
J Vet Med Sci. 2014 Nov;76(11):1471-8
[PMID:
25056673]
J Virol. 2013 Apr;87(8):4384-94
[PMID:
23388721]
Am J Trop Med Hyg. 1960 Jan;9:62-9
[PMID:
14410617]
PLoS Negl Trop Dis. 2020 Oct 28;14(10):e0008856
[PMID:
33112863]
Virology. 2010 Sep 1;404(2):139-47
[PMID:
20553924]
Bioessays. 2011 Feb;33(2):103-10
[PMID:
21140470]
Methods Mol Biol. 2018;1836:159-183
[PMID:
30151573]
Nat Rev Microbiol. 2014 Oct;12(10):673-85
[PMID:
25198140]
Cell Host Microbe. 2010 Jun 25;7(6):488-99
[PMID:
20542252]
PLoS Pathog. 2018 Jan 19;14(1):e1006845
[PMID:
29352299]
Virology. 2008 Aug 15;378(1):21-33
[PMID:
18554681]
Virology. 1997 Aug 18;235(1):40-7
[PMID:
9300035]
J Virol. 1976 Sep;19(3):985-97
[PMID:
972436]
Virus Res. 2008 Dec;138(1-2):139-43
[PMID:
18840482]
N Z Vet J. 2013 Mar;61(2):63-7
[PMID:
23215779]
Virology. 1993 Apr;193(2):993-6
[PMID:
8460503]
J Biol Chem. 2016 Feb 12;291(7):3411-22
[PMID:
26677217]
Cell Host Microbe. 2011 Jul 21;10(1):75-88
[PMID:
21767814]
J Virol. 2016 Jul 11;90(15):6784-98
[PMID:
27194760]
Curr Opin Virol. 2011 Jul;1(1):35-43
[PMID:
22440565]
J Virol. 2007 Dec;81(24):13378-84
[PMID:
17928356]
J Virol. 2017 Jul 12;91(15):
[PMID:
28539443]
J Virol. 2012 Aug;86(15):7988-8001
[PMID:
22623766]
EMBO J. 2011 Aug 31;30(17):3481-500
[PMID:
21878991]
J Virol. 2014 Aug;88(15):8565-78
[PMID:
24850728]
Virology. 2015 May;479-480:498-507
[PMID:
25866377]
Emerg Infect Dis. 2020 Jun;26(6):1287-1290
[PMID:
32441621]
Viruses. 2019 Mar 26;11(3):
[PMID:
30917612]
Viruses. 2016 May 24;8(5):
[PMID:
27213430]
Virology. 1982 Oct 15;122(1):191-7
[PMID:
7135833]
J Biol Chem. 2004 Jul 30;279(31):32035-45
[PMID:
15166245]
