Adenovirus 5 Vectors Expressing SARS-CoV-2 Proteins

Abstract

SARS-CoV-2 coronavirus is a recently identified novel coronavirus that is the causative agent of the COVID-19 pandemic that began in 2020. An intense research effort has been undertaken by the research community in order to better understand the molecular etiology of this virus and its mechanisms of host cell subjugation and immune system evasion. To facilitate further research into the SARS-CoV-2 coronavirus we have generated adenovirus 5-based viral vectors that express SARS-CoV-2 proteins-S, N, E, NSP7, NSP8, and NSP12 as hemagglutinin (HA)-tagged and untagged variants. We have also engineered two additional viruses that express the S protein receptor binding domain and a fusion of the receptor binding domain to the N protein. We show that these vectors are expressed in several different cell lines by Western blotting and real-time quantitative reverse transcriptase (qRT-PCR), we evaluate the subcellular localization of these viral proteins, and we show that these coronavirus proteins bind to a variety of cellular targets. The flexibility of adenovirus vectors allows them to be used in a variety of cell models and, importantly, in animal models as well. IMPORTANCE The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has brought untold personal and economic suffering to the world. Intense research has made tremendous progress in understanding how this virus works, yet much research remains to be done as new variants and continued evolution of the virus keep shifting the rules of engagement on the pandemic battlefield. Therefore, wide availability of resources and reagents to study SARS-CoV-2 is essential in overcoming the pandemic and for the prevention of future outbreaks. Our viral vectors provide additional tools for researchers to use in order to better understand the molecular biology of virus-host interactions and other aspects of SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; adenovirus; viral vectors.

FIG 1

Schematic of Cre-LoxP-based viral recombineering to generate rHAdV vectors. Diagram representing the recombineering strategy for making rHAdV vectors. Modified from reference with permission.

FIG 2

Viral protein expression in 293 and A549 cells infected with the indicated viral constructs. (A) 293 cells were infected with the indicated viruses at an MOI of 500 vp/cell; 24 h after infection cells were harvested and lysed. Then, 30 μg of the total cell lysate was resolved by SDS-PAGE and probed for HA using the rat monoclonal anti-HA antibody. Two exposures are shown, along with the molecular weight marker indicators. The predicted molecular weights in kDa for these proteins are as follows: NSP7, 9.4; NSP8, 22; NSP12, 107; E, 8.4; N, 46; RBD, 31; RBD-N, 76. (B) The same as panel A except A549 cells were infected at an MOI of 1,000 vp/cell for 72 h. (C) The same as panel B except cells were infected with untagged Ad.S at an MOI of 1,000 vp/cell for 72 h and blotted with anti-S antibody 2B3E5. #1 is a construct with a WPRE, whereas #2 does not have this element. The predicted molecular weight of S is 141 kDa.

FIG 3

Expression of nontagged SARS-CoV-2 mRNAs encoding SARS-CoV-2 proteins. 293 cells were infected with the indicated rHAdVs at an MOI of 10 vp/cell for 24 h. Total RNA was subsequently extracted using the TRIzol method, and cDNA was transcribed using VILO master mix. Real-time qRT-PCR was performed using primers specific for each gene and SYBR master mix for CFX on a Bio-Rad CFX96 real-time PCR instrument. GAPDH was used as an internal control. Error bars represent the standard deviation of 3 biological replicates. Numbers represent the percentage of GAPDH level.

FIG 4

SARS-CoV-2 proteins bind cellular proteins. rHAdV vectors expressing the indicated proteins were infected into 293 cells at an MOI of 100 vp/cell for 24 h. Cells were then harvested, lysed, and immunoprecipitated using anti-HA resin. Proteins were eluted and separated by SDS-PAGE on a 10% polyacrylamide gel, followed by silver staining. HA-tagged SARS-CoV-2 proteins are indicated with asterisks.

FIG 5

Subcellular localization of SARS-CoV-2 protein expressed from rHAdV vectors. HT1080 cells were infected with the indicated viruses at an MOI of 1,000 vp/cell. Then, 24 h after infection, cells were fixed and stained either for HA using the rat monoclonal 3F10 or for S using 2B3E5. DAPI was used as a nuclear counterstain, and representative images for each protein are shown. Images were acquired on a Zeiss LSM700 laser confocal microscope using a 63× lens objective.