Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-062-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Jul 31, 2023
Objective:
This research project is aimed at understanding viral-host interactions associated with Vesicular Stomatitis Virus (VSV) emerging strains. This understanding will provide scientific evidence to generate models that predict future outbreaks and to develop intervention strategies to minimize the impact of future disease outbreaks.
A library of single-gene Knock-Out Vero cells will be developed and used as a sceening tool against emerging strains of VSV. These cells will identify host genes involved in VSV infection. The cells will be generated though Clustered Regularly Interspeced Short Palidromic Repeats (CRISPR)-Cas9 technology. Following viral infection, the remaining viable cells are subjectd to next generation sequencing in order to identify which host genes are critical for virus-host interaction and virus infection. The intended goal of the proposed work is to identify host proteins involved in virus replication which could be targets for antiviral therapy.
Approach:
To generate the genome-scale CRISPR Knock-Out (GeCKO) Vero cells for screening, the lentiviral vector GeCKO v2 system which consists of a pooled library of 122,417 unique single guide RNAs (sgRNAs) targeting multiple conserved regions of 19,052 human genes will be used. The Cas9 nuclease, sgRNA, and antibiotic resistance gene for selection, are all incorporated into a lentivirus vector which can then be introduced to the cell line of interest, ie Vero cells. The antibiotic resistance gene is used for selection of only those cells which successfully incorporate the lentivirus. Sequencing is used to identify the gene knockouts. At this point, the generated GeCKO cell line consist of a mixture of cells carrying single-gene knockouts which can be used for screening studies.
Vero cells will be used for generating the GeCKO library, as they are permissive for VSV as well as a number of other viruses which can be adapted to the screening process. Although the human sgRNA library may not be fully compatible with Vero cells derived from African green monkey due to genetic variations, we anticipate to have adequate coverage due to the multiple (3-6) sgRNAs targeted to conserved regions of each of the 19,052 genes represented in the library. Once gene targets are identified, subsequent knockout studies can be performed in Vero cells or other cell types of interest with sgRNAs designed specifically for a respective host gene.
The generated GeCKO Vero cells will be used to screen for genes critical for viral infection, in this case, VSV. This is done by several rounds of selection by virus infection of the GeCKO Vero cells. Only cells that are resistant to lytic VSV infection remain viable and propagate. These surviving cells are subsequently sequenced to identify the knock-out genotypes of the surviving cells.
To confirm that the identified host genes/ proteins (gene knockouts) are critical for VSV replication, specific small/ short interfering RNA (siRNA) will be used to temporarily knock-down expression of each of the genes identified in the original surviving wildtype cell line. Vero cells will be infected and virus replication evaluated by expression of viral genes, proteins and/ or infectious progeny. This analysis will allow us to determine the effect of identified genes on VSV replication. Then, a more detailed analysis of the virus-host protein interaction can commence. Once a gene is identified and confirmed to be critical for VSV infection, in future studies, the CRISPR-Cas9 system can also be used to generate stable single-gene knockouts in Vero or other cell types or animals such as mice and pigs, for further study.
