In these days of virus pandemic and the rapid development of effective vaccines, is important to remember that human immunodeficiency virus type 1 (HIV-1) remains one of the major global threats to human health, with no cure or vaccine available.
As many other viruses, HIV-1 is potently inhibited by interferon (IFN). IFNs are a family of cytokines which work as an alarm system, letting know the cell that something bad is happening. Very much like the Gondor military mobilising by the sound of the horn of Gondor in the Lord of the Rings books, IFN-stimulated genes (ISGs) are the response to the IFN alarm signal. This array of hundreds of genes create a hostile environment for pathogen replication, with the final goal of halting it altogether.
HIV-1 is inhibited by a number of ISGs, including tetherin, TRIM5a or MX2. MX2 is a recently(ish) discovered inhibitor of HIV-1 (2013). While previously thought to be a rather irrelevant ISG, since its description as an HIV-1 restriction factor, it hasn’t stopped collecting enemies. There is now evidence for MX2 as an inhibitor of HIV-1, herpesviruses and hepatitis B and C viruses. While much is still unknown about the HIV-1 inhibitory mechanism of MX2, there are some pieces of evidence worth noting:
- MX2 binds to the viral capsid lattice, and this is necessary for viral inhibition.
- MX2 localizes to the nuclear envelope, where it interacts with nuclear pore proteins to inhibit HIV-1.
- The amino-terminal domain (NTD) of MX2 carries all the determinants necessary for viral inhibition, including the aforementioned capsid binding and nuclear envelope accumulation.
- A stretch of 3 arginines within the MX2 NTD (positions 11, 12 and 13) are essential for anti-viral activity and nuclear pore protein interaction.
Based on these premises, we designed a proteomics-based experiment, seeking to identify MX2 NTD interactors in general and triple arginine motif binders in particular. To our delight, 2 proteins emerged as very specific binders: PPP1CB and MYPT1, both members of the myosin light chain phosphatase (MLCP) holoenzyme. We found that these 2 proteins bind to the NTD, where they dephosphorylate serines that border the arginine-motif at positions 14, 17 and 18. Very interestingly, phosphorylation of these serine motif results in an inactive MX2, since this protein is unable to bind to the viral capsid.
Therefore, we found ourselves in kind of a pickle: PPP1CB and MYPT1 are necessary for MX2’s anti-viral activity because they are needed to dephosphorylate these 3 serines, but, why is an ISG like MX2, which it’s supposed to act only when a virus is infecting the cell, being phosphorylated (and consequently “switched off”) on the first place? Wouldn’t it be more reasonable just not to phosphorylate it, having the protein always ready for action?
The answer to this question came with the aid of some extremely thorough work by Melissa Kane, from Paul Beiniasz’s lab. In their studies they showed that MX2 inhibits the nuclear import of some non-viral cargo. Kindly, they provided us with their plasmid constructs, and with them we found that the phosphorylated (non-antiviral) form of MX2 is also debilitated for the inhibition of nuclear import. On top of this, we also found that IFN treatment reduces the level of phosphorylation of this serine motif, thereby promoting antiviral activity.
The identification of the IFN role on MX2 phosphorylation was only possible because we had the right tool to differentiate phosphorylated from unphosphorylated MX2: a specific antibody that only recognizes the phosphorylated serine motif. I should say that we did obtain this antibody besides my, let’s say, unsupportive opinion. Mike was keen in custom-making it, using a phosphomimetic peptide, but in my opinion, if mass spectrometry struggled to identify these positions, there wasn’t a chance a dirty antibody for some unlucky rabbit serum would do it. To my surprise (and Mike’s ever-lasting delight), the antibody worked like a charm.
While we don’t know yet what triggers this phosphorylation/dephosphorylation process, we propose a model where basal levels of MX2 are “turned off” by NTD phosphorylation, consequently avoiding inhibition of nuclear import. On the other hand, in the presence of IFN, not only is MX2 transcription upregulated, but the protein is “turned on” by NTD dephosphorylation. Therefore, making another analogy, we could think of MX2 as a pistol the cell has to fight HIV-1 infection: while you don’t need to shoot the virus, you keep the lock (phosphorylation) on the gun, so you don’t shoot your own foot. But, when is time to fight infection, the lock goes off and the pistol is ready.