HIV integrase compacts viral DNA into biphasic condensates

Reviewer #1 (Public review):

Summary:

The authors investigate the compaction of HIV DNA by the viral enzyme integrase (IN) in vitro.

Strengths:

The authors employ robust techniques, including single-molecule force microscopy and spectroscopy, to investigate the impact of IN-DNA interactions on DNA conformation. Additionally, they interpret their experimental findings using coarse-grained Monte Carlo simulations.

Weaknesses:

The authors could provide a more in-depth discussion of the biophysical reasons behind their experimental observations. Currently, there is insufficient analysis to explain why certain behaviors are observed experimentally.

Reviewer #2 (Public review):

Summary:

This is a high-quality biophysical study providing valuable new in vitro information on the modes of HIV-1 integrase protein (IN) interaction with the double stranded (ds)DNA.

Strengths:

Both main experimental approaches used in this study: magnetic tweezers (MT) and atomic force microscopy (AFM) are used at the state-of-the-art level.

Weaknesses:

(1) The findings of Fig.1 suggest modest preference of IN oligomers for the processed DNA ends typical of the viral dsDNA in the intasome and the DNA with blunt ends relative to the IN-oligomer binding to the random internal sites on DNA. This is an impressive result. Is it completely new? What was known about it? Can IN oligomer bind and unbind on the time of experiment? Is it an equilibrium preference? Was the effect of Mg2+ in that binding known?

(2) Regarding the AFM-observed IN-induced DNA bending and looping. How defined is the DNA crossover angle in the looped state? How many IN molecules typically hold it together? What density of IN per DNA length is needed to observe formation of IN oligomers, and their induced DNA beds and loops? It looks like more information on the two dsDNA crossover points held together by IN oligomers can be obtained from the AFM images, similar to the ones in Fig. S22. In particular, the preferred crossover angle (similar to bending angel of one DNA) and the total number of IN proteins within the oligomer holding this crossover point together can be extracted from the AFM data at higher resolution.

(3) Similarly, questions for Fig.3. What is the typical binding density (i.e. IN per DNA unit length) required for the IN-induced rosette formation? For the IN-induced 3D condensation? I understand that the AFM is not the good method to estimate the protein:DNA stoichiometry, as the mica surface and its treatment affect the protein/DNA interactions compared to the bulk solution. But still, in combination with the MT data there should be at least approximate estimate of the degree of DNA saturation. With IN oligomers that cause these sharp cooperative structural transitions of the complex. The fact that higher salt increases critical concentration of IN for these transitions is consistent with the critical levels of DNA saturation with IN required for each transition. Also, the fact that the rosette formation is not observed on shorter 3Kbp DNA but is observed on longer 4.8Kbp and 9Kbp comes from the lower probability of looping in the shorter DNA and can be discussed/interpreted. Maybe the persistence length of the DNA/IN complex at this level of its saturation can be estimated from these data. This persistence length should be shorter than for the bare DNA, as the IN binding induces DNA bending.

(4) In the section describing the simulations of the IN-induced dsDNA compaction the authors introduce a very simple model in which IN tetramer is presented as a bead of the size of ~12 bp similar to the binding site size of the singe IN on DNA with the four binding sites for DNA. It would be useful to discuss the published experimental structural data on the IN-DNA complexes available to better rationalize this choice of the model. In general, more overview of the available information on IN-DNA complexes and discussion of how present results fit into the general story and add to it would be useful. The authors fit their modeling results to their experimental data to obtain the individual monomeric IN-DNA interaction strength of 5 kBT. What is the geometry of these for DNA binding sites on the IN tetramer? Is it important for the complex structure? Also, the authors mention that the additional IN-IN interactions are required to reproduce their AFM results. What is the geometry and the strength of these interactions? It should matter for the structure of the IN-DNA aggregate. For example, if the IN molecules or DNA-bound oligomers were only interacting head-to-tail on the DNA that they bind to, it would lead to the filament formation, rather than the 3D condensate. What was the density of the IN oligomers on DNA to lead to each of the two AFM-observed transitions: (i) the "rosette formation" and (ii) the denser 3D aggregate formation? It may be possible to answer these important questions based on the AFM images. Is the higher resolution AFM measuring the oligomer sizes and their densities on the DNA possible?

(5) Regarding the elastic and viscoelastic properties of the IN-DNA complexes studied in Fig. 4. These are very interesting observations that could take more interpretation. For example, why is the rosette center in Fig.4C has lower stiffness that the loop area? Is it because in the loops the stiffness is more of the background and bare DNA is felt? Does the stiffness of the fully compacted complex in Fig.4D follow the density of the globule?

(6) Also, more interpretation of the observed dwell times and velocity distributions of the complex unfolding vs force can be provided, and what it tells us about the interactions that hold this complex together.

(7) The effect of ALINIs on the structure of rosette and denser condensate is interesting. Based on the published notion on where ALINIS bind to IN and what kind of interactions they prevent can these results be better interpreted? Maybe the IN-IN interactions that hold the rosette together are the same as the ones that hold the dense aggregate together, but just at higher [IN]? And because the fewer IN interactions have to hold large DNA loops in the rosette, they are weaker interactions that are easier to disrupt via the same ALINI-IN interactions?

(8) Finally, in the discussion it would be quite valuable if the authors could comment on the conclusions based on their findings for the in vivo IN-DNA interactions inside the mature capsid. As there are 100-150 IN molecules per capsid within the very small capsid volume, do all of these IN bunch up together on the dsDNA being synthesized? By the end of the reverse transcription when the vDNA ends are synthesized and processed, can this IN oligomer be re-bound to form the synapse of the vDNA ends?

Reviewer #3 (Public review):

Summary:

In this work, the authors aims and efforts point towards evaluating the interaction mechanisms between viral protein integrase (IN) and viral DNA. They develop a multifaceted approach to probe the effect that IN has on the formation and structure of IN-DNA complexes under different environmental conditions to determine the role of IN in early stages of infection. HIV infection is considered a global pandemic with huge challenges in both treatment and prevention. This work presents a step towards understanding the mechanisms in early infection and thus prevention.

The experimental work is carried out using single molecule imaging and force spectroscopy, alongside computational verification using Monte-Carlo simulations. The authors use a range of well-established methods to quantitatively evaluate this, pushing forward the current state of the art.

The paper shows that in the presence of IN, DNA is compacted into a condensate in a biphasic manner, first forming a 'semi-compact' rosette condensate followed by a fully compacted condensate. As HIV DNA must be fully compacted to enter the cell nucleus for infection, this work describes the importance of the role of IN and the conditions required for it to reach a full condensate, and hence provides a new understanding on the early role of IN in infection. Furthermore, the authors show that the semi-compact rosette condensate (i.e. the first phase) is susceptible to IN inhibitors whereas the second compaction phase is insusceptible. This work provides us with information that using inhibitors in the early stages of IN-DNA interaction, infection may be prevented.

Strengths:

The authors present a strong piece of work, using current experimental and computational methods to investigate IN-DNA interactions and to convincingly describe their experimental observations. Firstly the data and analysis shown from AFM and MT experiments convincingly show a two-phase compaction of DNA upon interaction with IN. The authors use Monte-Carlo simulations to model DNA-IN interactions, specifically showing that their experimental results of a two-phase compaction can only be observed via simulations if IN-IN attraction is included.

The authors aim of showing the effect of IN on the compaction of DNA was achieved successfully using AFM and MT. Furthermore, the works show clearly the susceptibility of the partially compacted DNA-IN core to inhibitors. Overall the conclusions in this paper are supported well by their experimental data and it is likely that this paper will not only be used as a model for future experimental work to explore other retroviral nucleoprotein condensation but also to develop a deeper understanding of the role of IN-inhibitors infection prevention.

Finally, the article is written very coherently and is well supported by critical analysis of their findings and appropriate referencing to supplementary figures.

Overall, this article is very worthy and through extensive and detailed work the authors probe difficult questions regarding HIV infection, which currently poses a huge global risk. The work completed by the authors substantially advances our understanding of HIV infection and can be used by those in the future to probe this question further.

Weaknesses:

Important aspects of the methodologies in this paper are not described in detail. For example, force volume curves have been used to evaluate the mechanical properties of the DNA-IN complex. Force-volume measurements are prone to a number of errors, particularly relating to data acquisition and analysis. The methodology presented is not clear on how the data is acquired, whether statically or in amplitude modulation, which affects analysis and interpretation. Although the authors do recognise some of the difficulties with force curve analysis, a more rigorous study could have been provided with citations to additional relevant literature (particularly taking note of the methods).

A minor point is that it is not clear that the AFM imaging is performed in air, in contrast to AFM force spectroscopy in liquid, which could affect the interpretation of the data and therefore comparisons which are drawn between the two. This is made more challenging as the methodology for the compaction measurements is not described in the methods, and the code is not provided. The source code should be made open-access and available to enable the work to be better understood and reproduced.