RR:C19 Evidence Scale rating by reviewer:
Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.
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Review: The authors of this manuscript show that under non-infected and infection conditions with HIV-1 or HSV the Mx2 protein co-localises with components of the nuclear pore or cytoplasmic ribonucleoprotein complexes. Downregulation of some of these accessory proteins impacts Mx2 ability to restrict HIV-1 and HSV entry into the nucleus.
Mx2 is an IFN-stimulated gene that serves as part of the anti-viral response. Mx2 is known to restrict the infection of some viruses including HIV-1 and HSV. Using a proximity-proteomics screen the authors define a Mx2 interactome consisting chiefly of previously identified binding partners. Under untreated, IFN-stimulated as well as infection conditions Mx2 forms two major interaction networks consisting of FG-signature nucleoporins (Nups) and granule-associated ribonucleoproteins. Due to the characteristics of these proteins, the authors hypothesize that they form biomolecular condensates together with Mx2.
They validate the functional role of 12 of these interactors by siRNA silencing. The gene knockdown of SAM4DA, TNPO1 and NUP62 leads to a subtle decrease in Mx2 dependent restriction of HIV-1 and HSV. Such effects might be more appreciable with gene knockout. The direct interaction of these three proteins with Mx2 is explored using NanoBIT assay. Binding of SAM4DA, TNPO1 and NUP62 to Mx2 is detectable but these interactions are relatively weak when compared to Mx2 dimerization. The sub-cellular localization of SAM4DA, TNPO1 and NUP62 with Mx2 is investigated using fluorescent live cell imaging, the co-localization of each partner to Mx2 is evident within large perinuclear condensates.
SAM4DA and TNPO1 knockdown differentially alter the size and arrangement of the Mx2-dependent condensates. By generating truncations or domain deletions of Mx2 they pinpointed the N-terminal domain as necessary for formation of condensate structures. A reduction in condensate formation also correlates with a lack of anti-viral activity. The binding of TNPO1 is also dependent on this domain. Deletion of the N-terminus causes a reduction in the localization of Mx2, but increase of HIV-1 at the nuclear envelope.
HIV-1 and HSV hijack microtubules to travel to the nucleus, Mx2 condensates also co-localize to the microtubules. Depolymerization of microtubules surprisingly does not disrupt Mx2 localization but does marginally decrease Mx2-mediated restriction of HIV-1. The reverse trend is observed with respect to HSV-1.
Overall the manuscript provides insights into the mechanism by which MxA restricts HIV-1 and HSV capsid entry into the nucleus. The authors highlight SAMD4A, TNPO1 and NUP35 as MxA interactors that comprise cytoplasmic condensates and speculate that these may function as nuclear pore decoys to divert incoming viruses. The overall contribution of these co-factors in anti-viral activity appears slight which could suggest a level of redundancy.
Note for authors:
It may be informative to directly compare the MxA interactome from untreated to infected - are some co-factors enriched during infection?
The paper is well written but very data heavy (with a lot of unseen data already in the supplementary).
Some of the conclusions from the infection experiments feel over-stated, for instance - the effects of SAMD4A, TNPO1, NUP35 in Figure 2A, B are not vastly dissimilar to the non-targeting control (judging from left hand panels). There also seems to be differing effects of KD (Nup58/62) when applying the HSV-1 and HIV-1 reporter systems. Overall SAMD4A knockdown appears insufficient. Perhaps complementing back with overexpression might give a more convincing effect.
Figure 4B would be better controlled with V5-TNPO1/SAMD4A/NUP35-SmBiT only plasmids to match the +Mx2-LgBiT experiments.
As written, figure 7 does not help strengthen the message of the paper.
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