SARS‐CoV‐2 Viral Budding and Entry can be Modeled Using BSL‐2 Level Virus‐Like Particles

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses is restricted to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus’ genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.

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The YLDL Sequence within Sendai Virus M Protein Is Critical for Budding of Virus-Like Particles and Interacts with Alix/AIP1 Independently of C Protein

Journal of Virology ◽

10.1128/jvi.02218-06 ◽

2006 ◽

Vol 81 (5) ◽

pp. 2263-2273 ◽

Cited By ~ 53

Author(s):

Takashi Irie ◽

Yukie Shimazu ◽

Tetsuya Yoshida ◽

Takemasa Sakaguchi

Keyword(s):

Matrix Protein ◽

Sendai Virus ◽

Specific Interaction ◽

M Protein ◽

Small Interfering Rnas ◽

C Protein ◽

Enveloped Viruses ◽

Virus Like Particles ◽

Viral Budding ◽

N Terminus

ABSTRACT For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.

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Structure of cellular ESCRT-III spirals and their relationship to HIV budding

eLife ◽

10.7554/elife.02184 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 80

Author(s):

Anil G Cashikar ◽

Soomin Shim ◽

Robyn Roth ◽

Michael R Maldazys ◽

John E Heuser ◽

...

Keyword(s):

Electron Microscopy ◽

Endocytic Pathway ◽

Multivesicular Bodies ◽

Virus Like Particles ◽

Functional Roles ◽

Aaa Atpase ◽

Escrt Machinery ◽

Viral Budding ◽

Hiv 1 ◽

Uniform Membrane

The ESCRT machinery along with the AAA+ ATPase Vps4 drive membrane scission for trafficking into multivesicular bodies in the endocytic pathway and for the topologically related processes of viral budding and cytokinesis, but how they accomplish this remains unclear. Using deep-etch electron microscopy, we find that endogenous ESCRT-III filaments stabilized by depleting cells of Vps4 create uniform membrane-deforming conical spirals which are assemblies of specific ESCRT-III heteropolymers. To explore functional roles for ESCRT-III filaments, we examine HIV-1 Gag-mediated budding of virus-like particles and find that depleting Vps4 traps ESCRT-III filaments around nascent Gag assemblies. Interpolating between the observed structures suggests a new role for Vps4 in separating ESCRT-III from Gag or other cargo to allow centripetal growth of a neck constricting ESCRT-III spiral.

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