1. Reviewer #3 (Public Review):

    In this study, the authors use a combination of fluorescent and electron microscopy to visualize the trafficking of HIV-1 viral particles during infection. The goal was to determine the components of nuclear HIV-1 virions during infection, and specifically to determine the degree to which reverse transcribed DNA in the nucleus associates with capsid protein and other fluorescent markers of infectious virions, such as fluorescently labeled integrase, which is increasingly used by many labs to track HIV-1 particles in the nucleus. The strengths of the manuscript lie in the imaging approaches used to quantitatively measure colocalization between viral DNA, CA and IN during infection, which are rigorous and well executed. Tomograms of high pressure frozen/plastic substituted samples showing apparently intact capsid cores at and within the nucleus are the most significant outcomes of the study and represent a significant technical achievement. These images provide some of the most compelling evidence to date that cores an enter the nucleus intact, despite previous studies suggesting that capsid disassembly occurs in the cytoplasm or at the nuclear pore.

    The weaknesses of the manuscript lie in the use of HeLa based target cells in all but one of the figures. Although the results in primary cells are generally consistent with the results observed in HeLa cells, differences between HeLa and primary cells have been noted in other studies, and the manuscript would have been significantly improved with more extensive use of primary cells throughout. Additionally, the numerous other recent recent studies that have demonstrated that reverse transcription and uncoating complete in the nucleus, including works from the Pathak, Diaz-Griffero, Di Nunzio, Dash and Campbell labs, among others, reduce the potential impact of these studies. The Di Nunzio lab, in particular, has recently published a nearly identical system for labeling the reverse transcribed HIV-1 genome during infection. However, differences in approach prevented that study from being able to conclude that intact capsids exist in the nucleus (or made such conclusions open to alternative interpretations). In contrast, the CLEM-ET studies in this manuscript unambiguously show intact cores in the nucleus, and this is an advance for the field, in addition to being a substantial technical achievement. Nevertheless, the prior studies in this area, published last year, do impact the novelty of the observations made in this manuscript.

    In the aggregate, this manuscript adds to a growing body of work suggesting that models of HIV-1 infection that have dominated the field for years should be reconsidered. As recently as 2 years ago, the idea that even a small amount of CA protein remained associated with the viral replication complex in the nucleus was somewhat heretical. While old models die hard, and some in the field are likely to debate how "intact" the capsid cores observed in this manuscript actually are, the idea that intact or nearly intact cores can enter the nucleus is increasingly difficult to deny in light of data provided here. This raises questions regarding how the HIV-1 core, which exceeds the generally accepted size limitation of nuclear pore complexes by 50%, based on the width of a capsid core, can enter the nuclear environment in an intact or nearly intact state (an issue that is addressed in the recent (and cited) Zila et al. bioRxiv paper, but not here).

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  2. Reviewer #2 (Public Review):

    Despite the fact that reverse transcription was discovered 50 years ago, there are still some black boxes regarding RT spatiotemporal activity. Recent studies elsewhere and here indicate that RT can occur in the nucleus, revising the "dogma" that RT occurs exclusively in the cytoplasm of infected cells. However, it is still debated whether this concept can be extended to all HIV target cells and which RT processes can start and finish in the nucleus. The authors also performed several experiments designed to show that uncoating (loss of capsid) occurs in the nucleus. The authors deserve credit for developing and applying complicated imaging technologies. However, live imaging data comes from pseudo-viruses, which have low infectivity, so high amounts of virus have been used to obtain some of the results. This is a limitation, and I have some reservations about the conclusions and the generalization of the results, and also about the lack of statistics for the CLEM-ET studies, probably owing to the complexity of the technique (detailed below). In addition, despite using state-of-the-art CLEM-ET, it is possible to visualize only structures with strong fluorescence and recognizable structures. I therefore wonder how can the authors can conclude that only the forms that still have a conical or partial conical shape are the most important to follow? It is possible that more flexible CA structures can access the nucleus and that the authors neglect them owing to limitations of the technology. Immuno-gold CA labeling could solve this issue, and the authors have the technologies required to perform these experiments.

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  3. Reviewer #1 (Public Review):

    The authors of this paper seek to understand how HIV infects cells. HIV is a retrovirus that harbors a core of RNA nucleic acid in complex with important replication enzymes such as reverse transcriptase. After infection, reverse transcriptase converts the RNA into DNA, which is then integrated into the chromosome. The authors used advanced imaging techniques to visualize the DNA that is made by reverse transcription. They used fluorescent readout markers of proteins to also look at the viral proteins that are brought into the cell and track with the viral DNA during the virus infection.

    From this work the authors conclude that reverse transcription is completed in the cell nucleus, that intact or nearly intact cores are the substrate for nuclear import, and that virus core uncoating likely occurs in the nucleus, immediately preceding the integration step. Moreover, by using electron tomography, they drill down to the sub-micron level to glean an ultrastructural view of the viral complexes that are performing these important HIV infection steps. Some of these complexes appear to be novel, and thus the work will be of interest to other scientists in this field.

    Weaknesses of the study include insufficient control samples for some of the experiments and also clarifying some of the approaches used and some of their interpretations of the data (detailed below). The authors of this paper could have also done a better job of citing papers published by other scientists who came up with very similar conclusions and/or used very similar techniques.

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  4. Evaluation Summary:

    The authors use a variety of complementary approaches to visualize and characterize events in the first half of the HIV life cycle, with some overlap between the latter studies and the recent (and cited) Zila et al. bioRxiv paper from some of the same authors. The data are generally of high quality, and many findings are in line with recent field advances indicating that reverse transcription completes in the nucleus, that intact/nearly intact cores are imported into the nucleus, and that nuclear uncoating likely occurs immediately prior to integration. The results provide the best evidence to date that intact capsids can enter the nucleus of target cells during infection, and will generally be of interest to the field, although the impact is diminished somewhat by similar recent publications from a number of other groups (including one case that used nearly identical labeling methods to follow viral complexes during infection). Issues that should be addressed include missing controls in some cases, some examples of over-interpretation and uneven citation, and the need for additional images to help bolster some of the claims. Strengths of the study include the rigorous characterization of infection using sophisticated imaging methods and, most importantly, the use of CLEM-ET to visualize viral capsids in the nucleus.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  5. The authors of the manuscript seek to better understand the mechanism by which HIV-1 uncoats capsid (CA) and integrates its own viral cDNA into the host genome. They hypothesize that the entire viral capsid along with the integration complex is required for proper viral integration. They accomplish this by infecting cells with VSV-G psuedotyped virus to deliver a viral particle that can be visualized from infection up until integration. Using live cell imaging, they show that largely complete HIV-1 capsid enters the nucleus of the cell and where then the viral cDNA is released by physical disruption of the capsid lattice. These findings corroborate with other very recent publications using structural techniques that show HIV-1 capsid partially disassembles via the HIV-1 integration complex. Moreover, the authors show that the mechanism is also conserved in primary cells which further strengthens the paper. Overall, the manuscript seeks to answer a very important question in the HIV field that remains poorly understood and is important for a clearer understanding of the viral lifecycle. The manuscript is well written and proposes an important hypothesis with results that clearly test it. By using an innovative viral delivery system, they were able to visualize the process of viral uncoating and integration, however, it could be strengthened by using a lower multiplicity of infection that is closer to an in vivo infection. The data contribute to the field's understanding of important HIV-1 biology and also brings about new questions about the viral lifecycle, such as the mechanism behind how the integration complex physically ruptures the viral capsid. As a whole, the manuscript itself is strong in its current state. The authors successfully test the hypothesis they set out to investigate and do so in a manner that provides further questions. The manuscript is publishable in its current state with minor revisions. The only major concern for the manuscript is that recently published structural studies have not been included in the citations and should be. I have a number of other constructive suggestions that may help to strengthen the paper. Major Comments/Essential Revisions: · In figure 2, a similar experimental setup from the first figure is used, however, the time point is changed to 24 hours without much reasoning. Using the same time point throughout the manuscript would provide a stronger foundation for experimental results. Or, perhaps, the authors can provide an explanation about why the time point was changed. · Figure 3 has an issue with a lot of background for IN SNAP.SiR imaging. The high amount of background for this marker should be cleaned up in order to properly assess the images and localization. This problem also persists in the quantification methods for this figure. Foci per cell were used to quantify this marker, however, in its current state it should not be. This was not considered a major problem because of the in vivo data shown later in the paper. However, the paper would be strengthened by improving the signal to background in this figure, if possible. If not, perhaps this should be mentioned as a caveat of the quantification.· Tomographic slices depicted in figure 4 are very difficult to discern. This could be improved by providing more slices or a clearer averaged image. If this is not possible, perhaps this panel could be moved to the supplementary material because it does not add much. The 3-D reconstructed image is beautiful and provides stronger support for the authors' claims.
    · Figure 5 would perhaps benefit from reducing the number of viral components that are present in the experiment. There are several very bright foci and the images themselves are difficult to distinguish cellular components. These images could perhaps be improved by reducing the viral components to a minimum as well as using several slices or clearer images. Or, at least, the EM image and the fluorescent photomicrographs could be shown individually as well as superimposed. Minor Comments: · Related to the last point above, throughout the manuscript, a MOI of 6 is used and results in several punctae on a per cell basis. Lowering the MOI to result in less punctae per cell could help better visualize the experiments and provide clearer pictures.
    · Figure 4 very nicely depicts the colocalization of several markers in the nucleus, however, it suffers from the same problems as previous figures where there are too many viral markers on a per cell basis. The experimental setup could benefit from reducing the number of viral components to the minimum required for visualization. · The final figure itself is very strong in that the images are much clearer to interpret because there are only a couple of foci per cell. The images themselves reproduce what was observed in previous figures but are in primary cells. These images could be improved by trying to clean up background, however, these images are how the authors should aim to represent images in the other figures.
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