Is uncoating required for completion of reverse transcription of HIV?

Question

The p24 capsid plays an important role in the replication of HIV, particularly in the reverse transcription and nuclear entry processes. The capsid protects the viral reverse transcription complex (RTC) from antiviral sensors of the host cells, and breaks apart to release the pre-integration complex (PIC) for integration. However, the precise timing of the uncoating is unclear. It’s generally accepted that HIV can initiate reverse transcription in intact viral capsids because the p24 hexamers can alternate between closed and open conformation, which opens up a tiny pore that allows nucleotides to get in and out. So the uncoating is likely to occur during or after reverse transcription. Some researchers speculate that the capsid is uncoated before the reverse transcription is finished. The reason is that the capsid is too small to accommodate the DNA product (however, the RNA template is hydrolyzed at the same time of reverse transcription, which means the volume of nucleic acids should remain the same), and double stranded DNA is more rigid than single stranded RNA. As the reverse transcription proceeds, the pressure builds up inside, which eventually ruptures the capsid. The rupture of the capsid also makes room for the reverse transcription to finish. So I come up with an interesting idea. If the uncoating of the capsid is mandatory for the completion of the reverse transcription, can we test it by using mutations or small molecules like lenacapavir to stabilize the capsid and prevent its rupture? If the reverse transcription is unable to finish inside intact capsids, the uncoating must occur before its completion.

Reference:

Jacques, D., McEwan, W., Hilditch, L. et al. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis. Nature 536, 349–353 (2016). doi.org/10.1038/nature19098

Zila, V., Müller, T. G., Müller, B., and Kräusslich, H.-G. (2021b). HIV-1 capsid is the key orchestrator of early viral replication. PLoS Pathog. 17:e1010109. doi: 10.1371/journal.ppat.1010109

Answer 1

At the time I couldn't find any info on this, but I recently came across a review covering the recent literature investigating this subject. To summarize, evidence currently supports entry of the intact capsid into the nucleus through the nuclear pore complex (NPC), (this is a surprising result!) reverse transcription occurring mostly in 'nuclear speckles', and capsid uncoating most likely occurs near the end of or shortly after cDNA transcript is complete.

The authors suggest the conical shape of the HIV capsid facilitates NPC entry, but there's no direct evidence for that yet.

Electron tomography and AFM studies directly observed partially broken capsids with nucleic acid exiting, both in cell nucleus and isolated capsids. This means there's likely no signal-mediated disassembly of the capsid and makes it more likely rupture is caused by internal stress.

Two observations that undercut the "cDNA > internal pressure > ruptured capsid" hypothesis are as follows:

However, the in silico model (130) also predicts that compaction of HIV-1 nucleic acids by the viral nucleocapsid (NC) protein, present within the core at high concentration, strongly alleviates outward forces acting on the capsid shell, and NC-mediated condensation of DNA into a tightly packed globule has been demonstrated in vitro (135). The limit of dsDNA length that can be accommodated within the HIV-1 capsid under authentic conditions is currently not known.

lentiviral vectors packaging RNA much smaller than 10,000 nucleotides have been shown to efficiently transduce cells and are thus capable of genome uncoating and integration.

OP seems to be familiar with the details of the subject, so I recommend reading the review in full if you have access.