SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles

Abstract

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 makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; 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. 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 makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; 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. Severe acute respiratory syndrome SARS-coronavirus 2 (SARS-CoV-2) emerged in December 2019 in Wuhan, China, and has since spread around the globe. As of late October 2020, the virus has been detected in 189 different countries and territories with more than 43 million confirmed cases and more than one million attributed fatalities (https://covid19.who.int). The virulence of coronaviruses has previously been observed in severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) outbreaks in the previous 2 decades; however, there still remains no FDA-approved treatment for any coronavirus. In order to develop therapeutics, the ability to study viruses must be accessible. Under current circumstances, the authentic live SARS-CoV-2 virus is restricted to BSL-3 containment facilities; while many of these facilities exist and have refocused their collective efforts on SARS-CoV-2 research, the greater scientific community could be vastly helpful in combating this pandemic if such research was BSL-2 compatible. For instance, BSL-2 models of other difficult-to-work-with BSL-3 and -4 pathogens such as SARS-CoV-1 (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar), MERS (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar), Ebola virus (3Noda T. Sagara H. Suzuki E. Takada A. Kida H. Kawaoka Y. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP.J. 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Boisen M.L. Muncy I.J. Magliato S.A. Henderson L.A. Schoepp R.J. Cashman K.A. Hensley L.E. Garry R.F. et al.Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.Virol. J. 2010; 7: 279Crossref PubMed Scopus (68) Google Scholar) have been implemented in the form of virus-like particles (VLPs). Thus, the development of BSL-2 compatible models and assays to study SARS-CoV-2 assembly, budding, and entry, as well as evaluate potential therapeutics, is imperative. In this work, we aimed to develop morphologically and functionally relevant BSL-2-compatible VLPs to model SARS-CoV-2 budding and entry. SARS-CoV-2 has a positive-sense single-stranded RNA genome of 29.7 kilobases, which shares 79.6% sequence identity with SARS-CoV-1 (7Zhou P. Yang X. Wang X. Hu B. Zhang L. Zhang W. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (12486) Google Scholar). Both SARS-CoV-1 and SARS-CoV-2 utilize host cell surface receptor angiotensin-converting enzyme 2 (ACE2) to stimulate cellular uptake of bound viral particles (7Zhou P. Yang X. Wang X. Hu B. Zhang L. Zhang W. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (12486) Google Scholar). Trafficked through the endocytic system of the cells, SARS-CoV-2 is eventually released into the cytoplasm where it utilizes ten open reading frames (ORFs) to encode numerous nonstructural proteins and four structural proteins (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). As has been described for other coronaviruses, the four structural proteins are in ucleoprotein (N), membrane protein (M), envelope protein (E), and spike protein (S) and are presumed responsible for maintaining the structural integrity of the enveloped SARS-CoV-2 virion (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). The M glycoprotein of coronaviruses drives the assembly and formation of progeny viral particles from the endoplasmic reticulum–Golgi intermediary complex (ERGIC) and is the most abundant viral structural protein in the virion (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). M oligomerizes to create a protein lattice across ERGIC membranes and interacts laterally with S and E, the other two viral membrane proteins, which are integrated into the structural matrix of budding (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar, A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). The of in assembly and budding is it has been to be for assembly of SARS-CoV-1 viral particles and as a (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar, A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar, coronavirus protein is in two with different on assembly and the Virol. 2015; PubMed Scopus Google Scholar). The S protein coronavirus particles their (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). for viral assembly and formation in the of S is required for progeny viral particles to a host cell (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar). The structural is responsible for the viral RNA genome to the structural which it through with the of M in an (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar, A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). These the four structural proteins the assembly, and budding of progeny coronavirus particles (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). budding into the ERGIC progeny viral particles are released from the cell by (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). these have yet to be in the SARS-CoV-2 are to a of the four SARS-CoV-1 structural proteins in cell has been to which be and to study the of the virus (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar). of the viral life cycle such as assembly (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar), budding (2Wang C. Zheng X. Gai W. Zhao Y. Wang H. Wang H. Feng N. Chi H. Qiu B. Li N. Wang T. Gao Y. Yang S. Xia X. MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.Oncotarget. 2016; 8: 12686-12694Crossref Scopus (108) Google Scholar, Wang Severe acute respiratory syndrome coronavirus protein ability to virus-like particles when for the human virus 2008; PubMed Scopus Google Scholar), (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar, Wang Severe acute respiratory syndrome coronavirus protein ability to virus-like particles when for the human virus 2008; PubMed Scopus Google Scholar), and entry S. C. Y. M. is for the cell entry of 2016; PubMed Scopus Google Scholar) have been for these VLPs morphologically and functionally to authentic the viral are and be in a BSL-2 SARS-CoV-2 VLPs have been to M as the of viral formation R. M. Li J. P. Li N. of SARS-CoV-2 virus-like particles by 2020; 8: PubMed Scopus Google Scholar) and for vaccine development H. development to pandemic using in and Immunol. 2020; PubMed Scopus Google have yet to be to study SARS-CoV-2 entry the entry inhibitors of SARS-CoV-2 have been using coronavirus assays such as S. M. Wang C. W. Feng S. F. L. L. S. C. F. Y. S. et of SARS-CoV-2 by a spike protein a to membrane 2020; PubMed Scopus Google Scholar) L. S. X. L. N. L. S. P. Chan J. et of SARS-CoV-2 through 2020; PubMed Scopus Google Scholar). VLPs a and model of and viral entry in a BSL-2 Herein, we methods for and of SARS-CoV-2 previous study by et R. M. Li J. P. Li N. of SARS-CoV-2 virus-like particles by 2020; 8: PubMed Scopus Google Scholar) M was released into the of and cells of other viral structural this M, E, S in cells and VLPs and with we cells with 2 and with M was in the cell when The cellular was as and when M was in this is to the of et where was in the cell when be by et of a while we an and the have SARS-CoV-1 M is to be and the is to be for with which is we to the of M. with N M was in the cell and and of N on M was confirmed with with M to induce to membrane was with of viral structural protein cells. cells and in with M, E, S. with and with with and with a with with and to in from to N was in the cell and a was in the N was in the this was with this was to N is is detected in the was N protein which with as of N formation in cells was observed with These are to a study previously in SARS-CoV-2 cells are with N protein M. B. P. N. The global of SARS-CoV-2 2020; PubMed Scopus Google Scholar). was in the cell to M, is by N as it becomes in the cell when to et and previous work other coronavirus VLPs R. M. Li J. P. Li N. of SARS-CoV-2 virus-like particles by 2020; 8: PubMed Scopus Google Scholar, E. virus protein is to the complex and release of virus-like particles.J. Virol. PubMed Scopus Google Scholar, J. A. S. of coronavirus protein in membrane from cells and PubMed Scopus Google Scholar), when was it was in the by of the a with of the of cells to no in the membrane when with cells S was in the cell in the of cells from the membrane S trafficking to the membrane was confirmed by using of the viral structural proteins could we of structural proteins with M, as M is to be the major of assembly in coronaviruses (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar, A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar, R. M. Li J. P. Li N. of SARS-CoV-2 virus-like particles by 2020; 8: PubMed Scopus Google Scholar). of M N M and M S and VLPs M in the cell and when when with N M was in the cell and M release a of M. of these with M major in membrane however, M N and M S In M N cells, filamentous observed while in M S cells, the M N which produced a of The of to the M N system an of in assembly and of M N cells in membrane and VLPs the cell of the M N which observed in the all four structural proteins and with particles of in observed with a M N S cells by and numerous VLPs the cell the of is in with release of SARS-CoV-1 viral particles M. J. M. A. H. E. in cells late of 2004; PubMed Scopus Google Scholar) and on SARS-CoV-2 cells M. B. P. N. The global of SARS-CoV-2 2020; PubMed Scopus Google Scholar). with the M N S released the most VLPs These are to those of et where S to M however, in their all four structural proteins a in to In the S as two the a of S by host model assembly and budding, we a by S of a to other remains in the by which a with Y. A. as a for PubMed Scopus Google Scholar). For in of we with the other viral structural proteins M, and E. cells and for of system from the to the membrane of the system with the of The of the VLPs detected in of the the structural of the ERGIC by and and and E, VLPs to the of spike protein The VLPs in these are well the of SARS-CoV-2 protein form the of of the other viral structural fluorescently we with M, and in cells. into VLPs was confirmed with The into VLPs was by host these VLPs be entry the entry of the entry was cells with in while cells As an and from cells was and to cells to from cells to in cells. model viral entry using we the from the entry an entry was cells and with as previously described in assembly and budding the and the of the detected as in the of the in are to utilize the host endocytic to entry into cells (8Fehr A.R. Perlman S. Coronaviruses: an overview of their replication and pathogenesis.Coronaviruses Methods Mol. Biol. 2015; 1282: 1-23Crossref PubMed Scopus (153) Google Scholar). SARS-CoV-2 viral particles to with endocytic and associated membrane protein a with cells J. R. M. Wang X. Zhang H. Hu H. Li Y. Hu W. Wang M. a of is in SARS-CoV-2 in 2020; PubMed Scopus Google Scholar). particles more with than most particles are into the endocytic evaluate the entry of SARS-CoV-2 and it with of the live we the entry on cells with endocytic and for using the for to be to two with from for to for related are two with are to other to in J. PubMed Scopus Google Scholar). As from and cells to endocytic cells, which and for no and under any of these conditions. to cells, and with an of which was when with of cells to cells, and an of which was when with to cells, and an of which was when with the Thus, SARS-CoV-2 VLPs with and as previously for authentic SARS-CoV-2. These systems a for SARS-CoV-2 entry mechanisms in a BSL-2 As SARS-CoV-2 to it is we to of In this work, we the ability of viral structural proteins to VLPs and M was to of M with N S was the minimal for protein was to the must in viral assembly and this the for of the of of S M N M N cells M N S is the most efficient SARS-CoV-2 system for These are in to the of et R. M. Li J. P. Li N. of SARS-CoV-2 virus-like particles by 2020; 8: PubMed Scopus Google however, the the minimal efficient system for SARS-CoV-2 is by the SARS-CoV-1 study of SARS-CoV-1 VLPs the minimal for efficient was M of severe acute respiratory syndrome coronavirus RNA into virus-like particles is Virol. PubMed Scopus Google Scholar), while study the minimal system was M N (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar). et the most efficient system for SARS-CoV-1 was M N E. As for MERS and other coronaviruses such as and M was to be the minimal system for efficient (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar, H. M.C. assembly of particles by co-expression of viral envelope protein J. PubMed Scopus Google Scholar). N was to be in in this work we N drives the formation of filamentous in cells. These authentic live virus which detected the formation of in SARS-CoV-2 cells M. B. P. N. The global of SARS-CoV-2 2020; PubMed Scopus Google Scholar). is these progeny virus particles to and cells, which is by of viral particles released the of with N in the of cells, this N have we to in In this work, we for the first a model of SARS-CoV-2 viral entry in a BSL-2 SARS-CoV-2 and In with live virus with the and the late In work, we to entry and it to for viral uptake and entry is for evaluation of entry we to evaluation of SARS-CoV-2 entry to has been to in in cells with structural however, many such was on S. Zhang T. Zhang Y. Wang H. F. virus virus-like particles induce in PubMed Scopus Google Scholar). we have for the first of S protein assembly and budding as well as the formation and of from the presumed ERGIC In this research resources for other BSL-2 laboratories in the to and SARS-CoV-2 assembly, budding, and entry and on the four structural proteins, and drug of viral assembly, budding, entry The and a from of The was from The was a to by The was by viral structural proteins of the structural protein are in and from was a from of W. M.J. N. J. M. K. H. M. enzyme 2 is a receptor for the PubMed Scopus Google Scholar). cells in with and and conditions. using M and as by was in was and the was was to the for the was well and for on to cells in and for the cells in with of SARS-CoV-2 in using with M and for to cells in for the of VLPs are to as to methods described for the of SARS-CoV-1 VLPs (1Siu Y.L. Teoh K.T. Lo J. Chan C.M. Kien F. Escriou N. Tsao S.W. Nicholls J.M. Altmeyer R. Peiris J.S. Bruzzone R. Nal B. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.J. Virol. 2008; 82: 11318-11330Crossref PubMed Scopus (320) Google Scholar), SARS-CoV-2 VLPs from the of cells was from cells and with for was on of a using a and for and in a in of cells using and for on by a and for the was and the was in and by for protein of cell was to and was using the was as a to cell in M M with in a and on to a membrane which was for and for M with M N with N with and S with and as was as a cell by membranes with by in and membranes with was using and using an cells of in with using to the the cells by by in by and in and with the cells and with the for to cells with and with using For of N protein cell to the for of VLPs was to a with in one of in of and on a an of a of a of and on a to in with a of the on the of with cells to for was with structural protein and cells in for cells with M and in by the with M the of M and for with of and in the the with and for of the and on the the the in of SARS-CoV-2 SARS-CoV-2 and in cells. was from cells and with for of was well to cells in a cells using an for cells for 2 For this cells with and membrane with for and with the in of SARS-CoV-2 SARS-CoV-2 and in cells. was from cells and with for of was well to cells on in a cells using an for cells for 2 cells with in and on for and for all steps with and in of for 2 with of was to the cell and for a was and the cells by with for on two in and with in a for and with an of in 2 and in and 2 in with The with cells on with and face on with and in the for from the by in from the the face from the of cells using a and on a of VLPs was using the as described in et Y. A. as a for PubMed Scopus Google Scholar). the in of SARS-CoV-2 SARS-CoV-2 and in cells. cells with and in on for The on the was as described in entry and and on the the in SARS-CoV-2 in cells. was from cells and with for of was well to cells in which been previously with and and and cells using an for cells for 2 this cells with and membrane with for and with was using the of using to on the with in The majority of are the For the be by the The have no of with the of this In to all of the of this we to and for in maintaining cells and to the and for their facilities for of in this C. B. P. and R. S of and the C. B. E. A. R. S. and Y. S. the and C. B. P. the with and from all C. B. E. A. and R. S. for the and R. S. Research in this was by the of of the to R. under B. and R. and of under E. A. The is the of the and the of the of

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