Cellular Antivirus: How Foreign DNA is Hushed
Like a computer antivirus, our cells are constantly on the lookout for things that do not belong. Our body’s first line of defense, immune cells, circulate through the bloodstream and attack anything they perceive as different, but what happens when something foreign manages to avoid the immune system long enough to invade one of our cells?
Such is often the case with retroviruses, like the human immunodeficiency virus (HIV), which stealthily invade our cells to make more copies of themselves. These viruses convert their RNA genetic information into DNA, which they then integrate into our cell’s own DNA. This allows the virus to hijack the cell to make more copies of itself. But this viral integration process is not perfect. Often some pieces of viral DNA remain unintegrated in the cell nucleus. Intriguingly, rather than being expressed by the cell, this unintegrated retroviral DNA is silenced .
This silencing has frustrated scientists who hoped to take advantage of non-integrating retroviral DNA for gene therapy, a process by which new genetic information is given to patients who are missing a certain gene or have a defective copy of it. Because this DNA is silenced and the mechanisms that control its silencing are unknown, this avenue of treatment has proved difficult .
In a new study published just last month in Nature, Zhu et al describe the cellular machinery that control the silencing of unintegrated retroviral DNA, a discovery that holds the potential to improve gene therapy and our knowledge of our cells’ viral defense system.
Zhu et al took advantage of a Murine leukemia virus (MLV) which cannot integrate its DNA into the cells it infects and that also contains the genetic information for green fluorescent protein (GFP). Its DNA remains unintegrated in the cell nucleus and thus, is silenced. However, if silencing of this unintegrated DNA were relieved, the GFP gene would be expressed, causing the cells to glow green.
Using a genome-wide Crispr-Cas9 screen, Zhu et al asked which genes were required for silencing unintegrated DNA. They found that the genes NP220; members of the HUSH complex: MPP8, TASOR, and PPHLN1; and SETDB1, a histone methyltransferase, all showed increased green fluorescence when lost and thus are necessary components of the silencing machinery.
To examine the function of these proteins further, the researchers asked if they interact with each other in the cell and if they bind unintegrated DNA. They found that NP220 directly interacts with the HUSH complex proteins MPP8 and TASOR and that the identified proteins do in fact bind to unintegrated DNA. NP220 binds to DNA through its DNA-binding domain, and when NP220 is lost, HUSH and SETDB1 can no longer bind DNA, indicating that NP220 is the main player in bringing the other silencing components to the unintegrated DNA.
These identified components are necessary for silencing unintegrated DNA, but how exactly does that silencing occur? The researchers showed that when NP220, MPP8, or SETDB1 were lost, there was a significant decrease in the repressive epigenetic mark H3K9me3 on unintegrated DNA, indicating that these proteins are necessary for maintaining this repressive mark. They also found that if the histone deacetylases HDAC1 and HDAC4 were lost, histone H3 acetylation of unintegrated DNA increased as did its expression. The researchers pressed further and found that HDAC4 binds to the zinc finger motif of NP220, thus linking NP220 to the observed epigenetic silencing of unintegrated retroviral DNA.
Finally, the researchers asked about how specifically NP220 bound to unintegrated DNA. They found that when they mutated or removed the cytidine clusters on retroviral DNA, NP220 could not bind as well. Interestingly, they also found that in cells where NP220 could not bind to the unintegrated DNA, the virus replicated faster, suggesting that NP220 is a key mediator in the protection of our cells against MLV retroviral gene expression.
NP220, the HUSH complex, and SETDB1 are necessary for silencing unintegrated MLV retroviral DNA; however, the researchers also found that only some, but not all, of these same factors are important for silencing unintegrated DNA of other retroviruses such as HIV type 1 and Mason-Pfizer monkey virus (MPMV). This result indicates that a variety of different factors may be at play in silencing other unintegrated retroviral DNA.
This work sheds light on a process that has been observed but not understood for many years. With this new knowledge about how retroviral DNA is silenced, scientists will likely be able to improve gene therapy and ask new questions about our cell’s defense systems against invading retroviruses.
Original Paper: Zhu Y, Wang GZ, Cingöz O, Goff SP (2018). NP220 mediates silencing of unintegrated retroviral DNA. Nature, 564(7735):278-282. DOI: 10.1038/s41586-018-0750-6.
 Orzalli MH, Knipe DM (2014). Cellular sensing of viral DNA and viral evasion mechanisms. Annu Rev Microbiol, 68: 477-92. doi: 10.1146/annurev-micro-091313-103409.
 Schneider WM, Wu DT, Amin V, Aiyer S, & Roth MJ (2012). MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA. Virology, 426(2):188-96. doi: 10.1016/j.virol.2012.01.034.