Switching from KSHV latency to lytic replication

From the Geballe lab, Human Biology Division & Cancer Consortium

What if I told you that seven human viruses cause 15% of all human cancers? Scary, isn't it? Viruses cause cancer, but how? This is the question that Dr. Annabel Olson is trying to answer during her postdoctoral fellowship in Dr. Adam Geballe's lab. Of the oncogenic viruses, Dr. Olson is interested in Kaposi Sarcoma-associated herpesvirus (KSHV), which causes Kaposi Sarcoma (KS), a prevalent cancer in Africa. The life cycle of KSHV consists of latent and lytic replication phases. Latency, a dormant state of the virus, is characterized by the expression of a small number of viral genes. During lytic replication, the virus actively makes more copies of itself. In cancer cells, KSHV establishes a latent infection and less than 5% of the cells undergo lytic replication. “Dissecting out which signaling factors are involved in regulating the transition from latency to lytic replicative stages will help us understand how this virus is dysregulating signaling within the cell to cause disease,” said Dr. Olson. Since kinases are enzymes with critical roles in communication within and between cells, Dr. Olson used a pharmacology-based kinome screen followed by Kinase Inhibitor Regularization (KiR) analysis to uncover which kinases are important for signaling during KSHV latent to lytic replication switch. This screening approach was originally developed by Dr. Taran Gujral, an Associate Professor in the Human Biology Division, and adapted to the study of KSHV’s replicative switch. In the featured study, Dr. Olson identified several kinases that promote or restrict KSHV reactivation, including epidermal growth factor receptor kinases (HERs/ERBBs) known to be involved in breast, lung, gastric and other cancers. These findings are available on BioRxiv.

To study KSHV reactivation, Dr. Olson created a lytic replication indicator virus (KSHVLRI) by adding a DNA fragment to the KSHV bacterial artificial chromosome genome (BAC). The BAC genome encodes a Green Fluorescent Protein (GFP) that is constitutively expressed to identify latently infected cells and mCherry, a different fluorescent protein containing a nuclear localization signal (NLS), for quantifying the number of cells containing replicating virus. The KSHVLRI was introduced into iSLK cells (human renal carcinoma epithelial cells) that encode a doxycycline (Dox)-inducible KHSV replication and transcription activator (RTA). Using this system, Dr. Olson monitored latently infected cells and KSHV reactivation after the addition of lytic inducing agents.  

Dr. Olson used iSLK cells latently infected with the KSHVLRI and treated with DMSO (control) or a panel of 22 kinase inhibitors, along with lytic inducing agents. After 72 hours, reactivation was measured by counting positive red (mCherry expressing) cells per field and cell survival by quantifying green (GFP expressing) area per field. Using these data, KiR modeling was used to predict which kinases alter KSHV reactivation. Based on the KiR predictions, 13 top kinases that are likely to regulate KSHV reactivation were selected. The authors found that many of these kinases, including ERBB4 and MKNK2, reduced KSHV reactivation when knocked down, indicating that these kinases promote lytic replication. Because ERBB4 and MKNK2 share substrates and signaling pathways with related kinases, Dr. Olson tested whether knocking down these related kinases altered KSHV reactivation. Similar to knocking down ERBB4, knocking down ERBB2 and ERBB3 reduced KSHV reactivation. In contrast to MKNK2, knocking down MKNK1 did not reduce reactivation. Instead MKNK1 knock down enhanced lytic replication and therefore promotes KSHV latency. These results indicate that “members of this kinase family have opposing roles in regulating KSHV reactivation,” Dr. Olson explained. The researchers also investigated the role of ERBBs during the later stages of lytic replication. From these experiments they observed another case of opposing roles in which ERBB2, ERBB3, and ERBB4 promote lytic gene expression and ERBB1 inhibits it. These findings again highlight that members within the same kinase family can act in opposite directions to regulate the switch between KSHV latency and lytic replication. 

For the ERBB kinase family, ERBB2 is the preferred dimerization partner for the other ERBBs. As ERBB3 is catalytically inactive, it must partner with another ERBB to activate signaling. In control and ERBB2 knockdown cells, reverse-phase protein arrays were used to determine signaling dependent on ERBB2. The results from these experiments suggest that ERBB1 and ERBB2 form a heterodimer in latently infected cells, ERBB2 phosphorylates ERRB1 under this condition, but this dimer is possibly disrupted once the virus is reactivated, as supported by less ERBB1 phosphorylation in cells treated with lytic replication inducing agents. Additionally, the authors investigated the activation of downstream proteins of ERBB2, including cyclic AMP-responsive element-binding protein 1 (CREB1). From these studies, the researchers found that during KSHV lytic replication, ERBB2 presence is necessary for CREB1 phosphorylation. This is a new finding that adds to the upstream activation of CREB1, a transcription factor previously shown to promote the expression of KSHV lytic genes. Thus, ERBB2 promotes lytic replication in part by activating CREB1.

As a pro-latent factor, ERBB1 binds to ERBB2 during latency and restricts lytic gene expression. As lytic replication is induced, the ERBB1:ERBB2 heterodimer is disrupted and ERBB2 interacts with ERBB3, a pro-lytic factor.
As a pro-latent factor, ERBB1 binds to ERBB2 during latency and restricts lytic gene expression. As lytic replication is induced, the ERBB1:ERBB2 heterodimer is disrupted and ERBB2 interacts with ERBB3, a pro-lytic factor. Image provided by Dr. Olson

“Our findings demonstrate the importance of the HER/ERBB family of kinases and other kinases to regulate KSHV life cycle transition,” said Dr. Olson. The working model for HER/ERBB signaling during KSHV infection includes ERBB1 binding to ERBB2 during latency to restrict the expression of lytic genes. After lytic replication is induced, the ERBB1:ERBB2 heterodimer is possibly disrupted, resulting in ERBB2 interacting with another ERBB, likely ERBB3, to promote lytic replication. “Understanding the factors that regulate the virus replicative cycle is part of the groundwork necessary to achieve the ultimate goal of effective KSHV-specific cancer therapeutics,” Dr. Olson concluded. 

The spotlighted research was supported by grants from National Institutes of Health and the National Science Foundation. 

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Dr. Adam Geballe and Dr. Michael Lagunoff contributed to this work.

Olson AT, Kang Y, Ladha AM, Lim CB, Lagunoff M, Gujral TS, Geballe AP. Polypharmacology-based kinome screen identifies new regulators of KSHV reactivation. bioRxiv [Preprint]. 2023 Feb 1:2023.02.01.526589. doi: 10.1101/2023.02.01.526589.