Construction and Characterization of a Reporter KSHV Clone for Monitoring Lytic Infection

On Ki Grina Omaira Cheng

Authors:  On Ki Grina Omaira Cheng, Zsolt Toth

Faculty Mentor:  Zsolt Toth 

College:  College of Dentistry

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncovirus, which can cause Kaposi’s sarcoma, one of the most frequent cancers in AIDS patients. To date, we still do not have any KSHV-specific antiviral treatments, which can be in part due to the fact that we still do not fully understand how the viral genes of KSHV are regulated in infected cells. My research focuses on investigating the regulation of the promoter region of the KSHV gene called RTA, which is required for KSHV lytic infection including replication and virus production. To study the promoter of RTA, which controls RTA expression, I created a reporter KSHV virus. Using Bacterial Artificial Chromosome-based homolog recombination technology I inserted the red fluorescence protein mCherry-coding sequence before RTA in the viral DNA to create a recombinant KSHV clone expressing mCherry-RTA fusion protein. When the RTA promoter gets activated mCherry-RTA will be expressed, which turn infected cells red. Thus, using fluorescence microscopy I will be able to monitor RTA promoter activity by observing red fluorescence signal in cells. Using restriction enzyme digestions, gel electrophoresis and DNA sequencing I confirmed that I succeeded in generating a reporter KSHV virus encoding mCherry-RTA.

Poster Pitch

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Poster

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Dominika Burbul
Dominika Burbul (@guest_922)
1 year ago

Hello!
It’s Dominika from the Papp lab. I love your poster, great job! Do you plan to continue this project somehow following the end of USP?

Omaira Cheng
Omaira Cheng (@guest_2832)
Reply to  Dominika Burbul
1 year ago

Thank you! I am not part of USP, but will definitely continue this project!

Bernadett Papp
Bernadett Papp (@guest_2020)
1 year ago

Hi Omaira!

Interesting work! I particularly enjoy seeing your newly developed visual tool for virology research!

Have you tried to test your newly developed virus in other cell types as well? Thanks! (BP)

Omaira Cheng
Omaira Cheng (@guest_3646)
Reply to  Bernadett Papp
1 year ago

Hi Dr. Papp, thank you! No, we only tested it on the iSLK cell line, but it would be interesting to try infecting other cell lines that also have RTA transgene with this recombinant clone and to observe the expression of mCherry.

ZT
ZT (@guest_2168)
1 year ago

Hi Omaira, I have a question about Fig 5. Do you think you could also see mCherry signal earlier than 72 hours virus reactivation? Tx

Omaira Cheng
Omaira Cheng (@guest_7356)
Reply to  ZT
1 year ago

Hi ZT! No, mCherry is not observed when the virus is in the dormant stage. Once reactivated to the lytic stage, the fusion protein will express, but the initial signal can be weak in the window of 0 to 72 hours.

Gabriela Peguero Kushner
Gabriela Peguero Kushner (@guest_4708)
1 year ago

Lovely poster and video! Do you know if the introduction of mCherry and this fusion protein may have an effect on RTA function?

Best,

Gaby

Omaira Cheng
Omaira Cheng (@guest_6498)
Reply to  Gabriela Peguero Kushner
1 year ago

Thanks, Gaby! Good question! I don’t think this fusion protein interferes with or affects RTA function but rather acts as a reporting signal regarding RTA activity. But I will definitely look into this possible issue.

Lauren Roberts
Lauren Roberts (@guest_6352)
1 year ago

Hey Omaira!

Great job on your poster presentation! 🙂 Will you be planning to express your new recombinant RTA clone into BCBL1 cells (now that you have established at iSLK cell line)? Why did you choose iSLK cells to test your new recombinant clone? Thanks! Awesome work!

LR

Omaira Cheng
Omaira Cheng (@guest_7248)
Reply to  Lauren Roberts
1 year ago

Hi Lauren! Thank you! Yes, I plan to establish BCBL1 with this clone, which can be used in many other experiments 🙂 The reason of choosing iSLK cells is that it has RTA transgene, which can be induced by Doxycycline and NaB (Sodium Butyrate), that can reactivate the virus from latency to lytic infection, hence the fusion protein mCherry-RTA would be produced.