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A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2
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A versatile reporter system to monitor virus infected cells and its application to1
dengue virus and SARS-CoV-22
3
Felix Pahmeiera, Christoper J Neufeldta, Berati Cerikana, Vibhu Prasada, Costantin4
Papeb,c, Vibor Laketad,, Alessia Ruggieria, Ralf Bartenschlagera,d,e,#, Mirko Cortesea,#5
6
aDepartment of Infectious Diseases, Molecular Virology, University of Heidelberg,7
Center for Integrative Infectious Disease Research, Heidelberg, Germany8
bHCI/IWR, Heidelberg University, Heidelberg, Germany9
cEuropean Molecular Biology Laboratory, Heidelberg, Germany10
dGerman Center for Infection Research, Heidelberg partner site, Heidelberg, Germany11
eDivision <Virus-Associated Carcinogenesis=, German Cancer Research Center12
(DKFZ), Heidelberg, Germany13
14
#Address correspondence to:15
ralf.bartenschlager@med.uni-heidelberg.de16
mirko.cortese@med.uni-heidelberg.de17
18
Running title: A reporter system for SARS-CoV-2 and dengue viruses19
20
Keywords: SARS-CoV-2; dengue virus; live cell imaging; fluorescent reporter21
system; reporter cell lines; viral proteases22
23
Word count for the abstract: 19524
Word count for the text: 516925
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
dengue virus and SARS-CoV-22
3
Felix Pahmeiera, Christoper J Neufeldta, Berati Cerikana, Vibhu Prasada, Costantin4
Papeb,c, Vibor Laketad,, Alessia Ruggieria, Ralf Bartenschlagera,d,e,#, Mirko Cortesea,#5
6
aDepartment of Infectious Diseases, Molecular Virology, University of Heidelberg,7
Center for Integrative Infectious Disease Research, Heidelberg, Germany8
bHCI/IWR, Heidelberg University, Heidelberg, Germany9
cEuropean Molecular Biology Laboratory, Heidelberg, Germany10
dGerman Center for Infection Research, Heidelberg partner site, Heidelberg, Germany11
eDivision <Virus-Associated Carcinogenesis=, German Cancer Research Center12
(DKFZ), Heidelberg, Germany13
14
#Address correspondence to:15
ralf.bartenschlager@med.uni-heidelberg.de16
mirko.cortese@med.uni-heidelberg.de17
18
Running title: A reporter system for SARS-CoV-2 and dengue viruses19
20
Keywords: SARS-CoV-2; dengue virus; live cell imaging; fluorescent reporter21
system; reporter cell lines; viral proteases22
23
Word count for the abstract: 19524
Word count for the text: 516925
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
ABSTRACT26
Positive-strand RNA viruses have been the etiological agents in several major disease27
outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue28
virus and Zika virus that cause millions of yearly infections and spread around the29
globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current30
pandemic. The severity of outbreaks caused by these viruses stresses the importance31
of virology research in determining mechanisms to limit virus spread and to curb32
disease severity. Such studies require molecular tools to decipher virus-host33
interactions and to develop effective interventions. Here, we describe the generation34
and characterization of a reporter system to visualize dengue virus and SARS-CoV-235
replication in live cells. The system is based on viral protease activity causing36
cleavage and nuclear translocation of an engineered fluorescent protein that is37
expressed in the infected cells. We show the suitability of the system for live cell38
imaging and visualization of single infected cells as well as for screening and testing39
of antiviral compounds. Given the modular building blocks, the system is easy to40
manipulate and can be adapted to any virus encoding a protease, thus offering a high41
degree of flexibility.42
IMPORTANCE43
Reporter systems are useful tools for fast and quantitative visualization of viral44
replication and spread within a host cell population. Here we describe a reporter45
system that takes advantage of virus-encoded proteases that are expressed in infected46
cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization47
sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing48
for rapid detection of the infected cells. Using this system, we demonstrate reliable49
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
Positive-strand RNA viruses have been the etiological agents in several major disease27
outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue28
virus and Zika virus that cause millions of yearly infections and spread around the29
globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current30
pandemic. The severity of outbreaks caused by these viruses stresses the importance31
of virology research in determining mechanisms to limit virus spread and to curb32
disease severity. Such studies require molecular tools to decipher virus-host33
interactions and to develop effective interventions. Here, we describe the generation34
and characterization of a reporter system to visualize dengue virus and SARS-CoV-235
replication in live cells. The system is based on viral protease activity causing36
cleavage and nuclear translocation of an engineered fluorescent protein that is37
expressed in the infected cells. We show the suitability of the system for live cell38
imaging and visualization of single infected cells as well as for screening and testing39
of antiviral compounds. Given the modular building blocks, the system is easy to40
manipulate and can be adapted to any virus encoding a protease, thus offering a high41
degree of flexibility.42
IMPORTANCE43
Reporter systems are useful tools for fast and quantitative visualization of viral44
replication and spread within a host cell population. Here we describe a reporter45
system that takes advantage of virus-encoded proteases that are expressed in infected46
cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization47
sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing48
for rapid detection of the infected cells. Using this system, we demonstrate reliable49
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
reporting activity for two major human pathogens from the Flaviviridae and the50
Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter51
system to live cell imaging and use it for proof-of-concept to validate antiviral activity52
of a nucleoside analogue. This reporter system is not only an invaluable tool for the53
characterization of viral replication, but also for the discovery and development of54
antivirals that are urgently needed to halt the spread of these viruses.55
INTRODUCTION56
Positive sense single stranded RNA viruses constitute a major fraction of endemic and57
emerging human viruses (1). Among the positive-strand RNA viruses, flaviviruses58
such as dengue virus (DENV) and Zika virus (ZIKV) are some of the most prevalent59
arboviral pathogens and are considered a major public health problem (2, 3).60
Currently, there are no universal vaccines or specific antiviral drug approved for the61
prevention or treatment of infections with these viruses (4). Members of the62
Coronaviridae family also have a positive-strand RNA genome and have caused63
major outbreaks in the last two decades (5, 6). Currently, the world is facing the64
pandemic outbreak of SARS-CoV-2, the causative agent of coronavirus disease 201965
(COVID-19) (7, 8). As of August 2020, over 19 million confirmed cases and more66
than 700,000 confirmed deaths have been reported in 216 countries (9). Despite67
immense efforts by research teams around the world, there is still a dire need for68
effective and widely available treatment options and a prophylactic vaccine.69
Once released into the cell, the full genome of flaviviruses and the large open reading70
frame (ORF1ab) of coronaviruses are translated as polyproteins. Signal peptides and71
internal transmembrane regions direct polyprotein synthesis to the endoplasmic72
reticulum (ER) membrane where co-translational cleavage generates the mature viral73
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter51
system to live cell imaging and use it for proof-of-concept to validate antiviral activity52
of a nucleoside analogue. This reporter system is not only an invaluable tool for the53
characterization of viral replication, but also for the discovery and development of54
antivirals that are urgently needed to halt the spread of these viruses.55
INTRODUCTION56
Positive sense single stranded RNA viruses constitute a major fraction of endemic and57
emerging human viruses (1). Among the positive-strand RNA viruses, flaviviruses58
such as dengue virus (DENV) and Zika virus (ZIKV) are some of the most prevalent59
arboviral pathogens and are considered a major public health problem (2, 3).60
Currently, there are no universal vaccines or specific antiviral drug approved for the61
prevention or treatment of infections with these viruses (4). Members of the62
Coronaviridae family also have a positive-strand RNA genome and have caused63
major outbreaks in the last two decades (5, 6). Currently, the world is facing the64
pandemic outbreak of SARS-CoV-2, the causative agent of coronavirus disease 201965
(COVID-19) (7, 8). As of August 2020, over 19 million confirmed cases and more66
than 700,000 confirmed deaths have been reported in 216 countries (9). Despite67
immense efforts by research teams around the world, there is still a dire need for68
effective and widely available treatment options and a prophylactic vaccine.69
Once released into the cell, the full genome of flaviviruses and the large open reading70
frame (ORF1ab) of coronaviruses are translated as polyproteins. Signal peptides and71
internal transmembrane regions direct polyprotein synthesis to the endoplasmic72
reticulum (ER) membrane where co-translational cleavage generates the mature viral73
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
proteins (10, 11). The flaviviral protease NS2B/3, together with host proteases,74
cleaves the flavivirus polyprotein into three structural and seven non-structural75
proteins (12, 13). In the case of coronaviruses, ORF1ab is expressed as two76
polyproteins, which are cleaved into sixteen non-structural proteins (nsp) by the viral77
papain-like protease (PLpro) residing in nsp3 and the 3C-like protease (3CLpro) of nsp578
(14–17). The replication of viral RNA of both virus groups was shown to occur on ER79
derived membranes, in specialized virus-induced membrane compartments termed80
replication organelles (10, 11, 18–20).81
Reporter systems for detection of virus infection are an invaluable tool for the82
characterization and quantification of virus infection kinetics, for the characterization83
of virus-host cell interactions and for the identification of antiviral compounds. One84
approach is the insertion of tags into the viral genome that, upon replication and85
translation, allow for visualization of the infected cells. However, this approach86
requires functional molecular clones of a given genome, which are not always87
available. In addition, insertion of a tag frequently causes attenuation of viral88
replication competency and therefore, the search for adequate insertion sites is time-89
consuming or might fail.90
An alternative approach is the use of engineered fluorescent reporter proteins stably91
expressed in cells and altering their subcellular distribution upon viral infection (21–92
23). Building on this idea, here we established a reporter system based on an ER-93
anchored green fluorescent protein (GFP) that upon cleavage by a viral protease is94
released from the ER and translocated into the nucleus. Using this system, we95
demonstrate the reliable reporting activity of DENV and SARS-CoV-2 infected cells.96
Moreover, we apply this reporter cell system to live cell imaging and assessment of an97
antiviral compound.98
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
cleaves the flavivirus polyprotein into three structural and seven non-structural75
proteins (12, 13). In the case of coronaviruses, ORF1ab is expressed as two76
polyproteins, which are cleaved into sixteen non-structural proteins (nsp) by the viral77
papain-like protease (PLpro) residing in nsp3 and the 3C-like protease (3CLpro) of nsp578
(14–17). The replication of viral RNA of both virus groups was shown to occur on ER79
derived membranes, in specialized virus-induced membrane compartments termed80
replication organelles (10, 11, 18–20).81
Reporter systems for detection of virus infection are an invaluable tool for the82
characterization and quantification of virus infection kinetics, for the characterization83
of virus-host cell interactions and for the identification of antiviral compounds. One84
approach is the insertion of tags into the viral genome that, upon replication and85
translation, allow for visualization of the infected cells. However, this approach86
requires functional molecular clones of a given genome, which are not always87
available. In addition, insertion of a tag frequently causes attenuation of viral88
replication competency and therefore, the search for adequate insertion sites is time-89
consuming or might fail.90
An alternative approach is the use of engineered fluorescent reporter proteins stably91
expressed in cells and altering their subcellular distribution upon viral infection (21–92
23). Building on this idea, here we established a reporter system based on an ER-93
anchored green fluorescent protein (GFP) that upon cleavage by a viral protease is94
released from the ER and translocated into the nucleus. Using this system, we95
demonstrate the reliable reporting activity of DENV and SARS-CoV-2 infected cells.96
Moreover, we apply this reporter cell system to live cell imaging and assessment of an97
antiviral compound.98
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
MATERIALS AND METHODS99
Cell lines and virus strains. HEK-293T, A549 and VeroE6 cells were purchased100
from ATCC; Huh7 cells (24) were obtained from Heinz Schaller (Center for101
Molecular Biology, Heidelberg). Generation of the cell lines Huh7-Lunet and the102
derivative Huh7-Lunet-T7, stably expressing the RNA polymerase of bacteriophage103
T7, have been previously described (25, 26). All cells were cultured at 37°C and 5%104
CO2 in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) containing105
10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1% non-106
essential amino acids (complete medium). Huh7-Lunet-T7 cells were cultured in107
complete medium, supplemented with 5 µg/mL zeocin. A549-ACE2 were generated108
by transduction of A549 with lentiviruses encoding for the human Angiotensin-109
converting enzyme 2 (ACE2) gene as previously described (41).110
Wild-type (WT) DENV-2 was produced from an infectious molecular clone based on111
strain 16681 as described elsewhere (27). The DENV reporter virus genome encoding112
the Turbo far red fluorescent protein FP635 (DENV-faR) has been previously113
described (28). SARS-CoV-2 (strain BavPat1) was kindly provided by Prof. Christian114
Drosten (Charité Berlin, Germany) through the European Virology Archive. Except115
for DENV-faR that was generated by electroporation of BHK-21 cells as previously116
described (28), all virus stocks were generated by infection of VeroE6 cells.117
Supernatants were harvested, filtered, and virus concentration was determined by118
plaque assay. For infection experiments, cells were inoculated as specified in the119
results section for 1 h at 37°C. Fresh complete medium was then added, and cells120
were incubated for the indicated time spans.121
122
123
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
Cell lines and virus strains. HEK-293T, A549 and VeroE6 cells were purchased100
from ATCC; Huh7 cells (24) were obtained from Heinz Schaller (Center for101
Molecular Biology, Heidelberg). Generation of the cell lines Huh7-Lunet and the102
derivative Huh7-Lunet-T7, stably expressing the RNA polymerase of bacteriophage103
T7, have been previously described (25, 26). All cells were cultured at 37°C and 5%104
CO2 in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) containing105
10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1% non-106
essential amino acids (complete medium). Huh7-Lunet-T7 cells were cultured in107
complete medium, supplemented with 5 µg/mL zeocin. A549-ACE2 were generated108
by transduction of A549 with lentiviruses encoding for the human Angiotensin-109
converting enzyme 2 (ACE2) gene as previously described (41).110
Wild-type (WT) DENV-2 was produced from an infectious molecular clone based on111
strain 16681 as described elsewhere (27). The DENV reporter virus genome encoding112
the Turbo far red fluorescent protein FP635 (DENV-faR) has been previously113
described (28). SARS-CoV-2 (strain BavPat1) was kindly provided by Prof. Christian114
Drosten (Charité Berlin, Germany) through the European Virology Archive. Except115
for DENV-faR that was generated by electroporation of BHK-21 cells as previously116
described (28), all virus stocks were generated by infection of VeroE6 cells.117
Supernatants were harvested, filtered, and virus concentration was determined by118
plaque assay. For infection experiments, cells were inoculated as specified in the119
results section for 1 h at 37°C. Fresh complete medium was then added, and cells120
were incubated for the indicated time spans.121
122
123
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
100%
