Makram Mghezzi-Habellah

In the past, we showed that during HTLV-1 infections, the RNA decay pathway called NMD (Nonsense mediated MRNA Decay), plays an antiviral function, notably by targeting the unspliced viral RNA (gRNA). To protect gRNA, we characterized how the viral protein, HTLV-1 sequester the NMD helicase UPF1 in the nucleus, in a CRM1 dependant manner, leading to NMD inhibition. Alongside, CRM1 is an exportin demonstrated to be hijacked by Rex to export gRNA: Rex bind simultaneously to CRM1 and gRNA, while CRM1 drives the nuclear export of this viral RNP. Due to the common involvement of Rex and CRM1, we wonder if there is a coordination between NMD inhibition and gRNA export.

To address these matters, we performed protein immunoprecipitations in different HTLV-1 infected lymphocytes, expressing Rex or not. We found that the UPF1/CRM1 complex is strongly favoured/stabilized in the presence of Rex. To study its consequences for UPF1, we modelled UPF1/CRM1 interaction: first we precisely identified the NES domain of UPF1 before performing molecular dynamic simulations with coarse grain representation. These simulations suggest that when associated with CRM1, UPF1 may have a reduced affinity for RNA and UPF2, what matches with the observed NMD inhibition. To challenge these simulations more precisely, we performed RNA IP that corroborated a lower binding of UPF1 with endogenous RNA and UPF2, when Rex favours UPF1/CRM1 binding.

To the contrary, in the presence of Rex, UPF1 was more loaded on gRNA, driven by the specific binding of Rex to the RxRE motif of gRNA.  In this context, luciferase assays and fractionations showed that UPF1 was indeed needed to favour nuclear export of HTLV-1 gRNA. In the late steps of retroviruses replication cycle, gRNA is encapsidated in viral particles. Combining confocal microscopy, mass spectrometry and RNA IP, we also demonstrated that UPF1 is also incorporated in HTLV-1 virions, associated with gRNA. However, after UPF1 extinction, gRNA incorporation in viral particles, as well as GAG maturation, are significantly reduced, independently from NMD inhibition. Finally, by monitoring viral transmission between C91PL and uninfected Jurkat, we could correlate these deregulations with defects in virions infectiousness.

Altogether, our data demonstrate that NMD inhibition by Rex is coordinated with UPF1 hijacking and specific redirection towards gRNA. As a part of the gRNP, UPF1 supports viral functions during HTLV-1 replication cycle, ultimately favoring the viral transmission.