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We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes. 

Sandra Schifferdecker, Vojtech Zila, Thorsten G. Muller, Volkan Sakin, Maria Anders-Osswein, Vibor Laketa, Hans-Georg Krausslich, Barbara Muller. bioRxiv 2021.09.14.460218; doi: https://www.biorxiv.org/content/10.1101/2021.09.14.460218v2

We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: HIV capsid uncoating mechanisms remain poorly understood. Recent advanced microscopy studies have revealed intact HIV capsids in the cell nucleus, as well as transiting the nuclear pore complex (NPC). These studies revealed that the diameter of the NPC is greater than previously appreciated, and sufficiently wide to allow HIV capsid entry. While these advances in microscopy enabled remarkable visualization of these processes, studies to date remain limited by the ability to label and track HIV capsids, which are not very amenable to traditional methods of immunostaining or engineering fluorescent fusion proteins. Here, Barbara Muller’s group reports on the expansion of the genetic code of HIV to allow site-specific incorporation of a non-canonical amino acid into the capsid (CA) protein via amber suppression. HIV particles were treated with a membrane-permeable fluorescent molecule that reacted with the non-canonical amino acid in CA, rendering the particles intrinsically fluorescent. This fluorescent HIV was used to track HIV entry into a HeLa cell-based reporter cell line, as well as primary patient-derived T cells. These fluorescent HIV virions were slightly delayed in trafficking to the nucleus compared to parental HIV. High MOI infections demonstrated that HIV particles accumulated in clusters in the nucleoplasm. These capsids were largely intact, based on measurements of fluorescence intensity of particles in the nucleus compared to the cytoplasm, and the known stoichiometry of capsid-associated CA compared to total CA in HIV particles. Cryo-EM studies of infected T cells revealed that these largely intact HIV capsids in the nucleus and nevertheless somewhat deformed compared to canonical bullet-shaped capsids. This preprint clearly establishes the feasibility of HIV capsid labelling via genetic code expansion and takes important first steps towards characterizing fluorescent capsids in the nucleus. This technology should enable future mechanistic studies of HIV uncoating.   

OVERALL ASSESSMENT: This preprint represents a strong technical advance in the HIV field, which will enable future studies of capsid trafficking and uncoating. It will be particularly valuable when using advanced microscopy methods. Incorporating methods to track HIV genomes in the context of these studies will further advance the application of this technology. The manuscript is very well written, and most messages are clearly conveyed, although the reader would benefit from a more thorough explanation of GCE and capsid labeling methodology. Studies were generally well supported by appropriate numbers of biological and technical replicates, and statistical tests. Overall data quality was good, although some data that had been relegated to the supplemental material could be retrieved to the main figure set to support the authors’ conclusions more clearly.  

STRENGTHS:

-        Innovative approach and strong technical advance and starting place for future studies of HIV capsid trafficking and uncoating. 

-        Quantitative evidence for intact capsids in the nucleus based on CA*(SiR) signal intensity in the nucleus compared to the cytoplasm was viewed as a very clever approach that supports recent microscopy data in field. 

WEAKNESSES:   

-        Could be strengthened by a more fulsome explanation (with cartoons) of GCE and click-labelling methodology, and its application to HIV capsid labeling specifically. 

-        

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale) SCORE = 2

●      Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

o   Figure 1A: The GCE and capsid labeling methodologies really deserve a standalone figure of their own, and more explanation in the body text so the reader can fully appreciate how cool it is. Otherwise, it goes by too quickly. In class, we also discussed whether the Vpr amber stop codon was the only one in the HIV genome that needed to be changed to set up the CA ORF mutagenesis and GCE. Please clarify. A genome map marking relevant features including Vpr ORF and CA ORF could be helpful. The current cartoon image in Figure 1A is low-resolution, which also makes interpretation more difficult. 

o   Figure 1B: The representative TEM images could be improved by labelling features that the reader should notice. Images of budding viral particles are common in the HIV field, but general interest readers might not get everything out of the images that you want them to. 

o   Figures 1C/1D/1E: We wondered why there was lower yield of the HIV-1*CA14-SiR particles in Figures 1C/1D, but comparable or higher levels of viral structural proteins in Figure 1E. Please clarify. Also, please label the ~48 kDa additional CA band in the HIV-1*CA14-SiR lane in Figure 1E. 

o   Figure 2: Good. Minor point: some readers found the figure layout was a little difficult to follow at first and missed that Figure 2A is the quantitation for Figure 2A. Maybe consider revising layout for greater clarity.

o   Figure 3: Good. We wondered whether the particle quantitation in different cellular compartments in Figure 3D would be more informative in the form of violin plots as elsewhere in the manuscript. Were statistical tests performed on this data?

o   Figure 4: Good. We thought that the greyscale data in Figure 4C would be clearer if presented in colour.

o   Figure 5: There was considerable discussion about the use of the small molecule inhibitor PF74 as a control in Figure 5C. PF74 has been reported to interfere with capsid binding to host nucleoporins and the CPSF6 protein and reported effects range between capsid destabilization to having no effect on nuclear accumulation of capsids. Here, according to the figure legend, infected TZM-bl cells were treated with DMSO vehicle control or 15 uM PF74 for 1 h prior to fixation at 17 hpi. The result was that there was no significant effect on the nuclear accumulation of fluorescent capsids in the presence of PF74. However, the authors’ statement about PF74 displacement of CPSF6 from capsids was unsupported by the data provided. The PF74 treatment was quite brief at only 1 h prior to fixation, and the reader was not given any rationale for this drug dosage or timing. Overall, the data in Fig 5C could be strengthened by using a range of drug concentrations and validating the displacement of CPSF6 that was inferred by the authors. 

o   Furthermore, readers thought that the capsid cartoon in Figure 5A could be improved to provide a stronger link to the data in Figures 5B and 5C. The description of the excess CA proteins that were not incorporated into the capsid proper was surprising to some readers who were less familiar with HIV literature. Could elements of this cartoon be incorporated into a revised Figure 1 describing the GCE/click-labelling procedure in greater detail?     

o   Figure 6: In Figure 6A, claims that CPSF6 dissociation is enabling binding of antibody should be better supported. CPSF6 immunostaining could strengthen this point. At least the authors should discuss this if there are technical reasons why this was not done. 

o   Furthermore, one of our students who has some experience with Cryo-EM commented on the representative CLEM-ET data in Figure 6D-6E. They were hoping for more detailed descriptions of the methods. They also thought that whileisosurface rendering gave general structural information about capsids, subtomogram averaging could give more details about the capsid and may reveal whether they are truly largely intact, as indicated by the CA*(SiR) quantitation in Figure 5b. 

o   Minor point: white text on ET images is difficult to see. 

●      Are specific analyses performed using methods that are consistent with answering the specific question?

o   The experimental approaches were generally sound and addressed the specific questions. 

o   We thought that the experiments involving the PF74 drug needed better explanation and an approach that directly tested effects on CPSF6 binding, which was mentioned in the text. 

●      Is there appropriate technical expertise in the collection and analysis of data presented?

o   Appropriate technical expertise was demonstrated.

●      Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

o   Generally good throughout. Statistical comparisons were appropriate. 

●      Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

o   The experimental foundations of this study are generally solid. The authors cited the literature appropriately and presented data that largely supported their conclusions. The technical achievements described in the manuscript affirm other newly emerged data in the field regarding intact HIV capsids in the nucleus.  

2. Quality: Completeness (1–3 scale) SCORE = 1.5

●      Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

o   Generally, the data supports the conclusions as stated in the title and abstract. However, we thought that the conclusions of the experiments involving the PF74 drug needed better supporting data.

o   We also wondered whether the delayed nuclear entry of click-labelled capsids might be a limitation of this technology that should have been explored and discussed further. 

●      Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

o   N/A 

3. Quality: Reproducibility (1–3 scale) SCORE = 2

●      Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

o   Sufficient biological and technical replicates were performed to provide a high-confidence dataset.

●      Is there sufficient raw data presented to assess rigor of the analysis?

o   This was a weakness in the manuscript. While representative images were clear and supported the main findings, some excellent supporting data was buried in the supplementary file. We believe that the manuscript would tell a more compelling story if some of the strong supplementary data were retrieved and presented with the main figures. 

o   We also thought that there was insufficient raw data in the CLEM-ET experiments. 

Are methods for experimentation and analysis adequately outlined to permit reproducibility?

o   The methods are generally clearly described, but as mentioned above, more details and a detailed figure about the GCE and click labelling experiments would be very helpful for the general reader. 

●      If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

o   The experiments in primary T cells serve as a validation of the HeLa cell studies. 

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

●       Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

o    Yes

●       Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

o    Yes, but a more detailed coverage of the GCE/click-labelling as requested may necessitate more citations from the literature. 

●       Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

o    Overall, the paper was very well written and ideas were clearly conveyed, except in situations noted above, and repeated here: 

o    Figure 1A: The GCE and capsid labeling methodologies really deserve a standalone figure of their own, and more explanation in the body text so the reader can fully appreciate how cool it is.

o    Figure 1B: The representative TEM images could be improved by labelling features that the reader should notice.

o    Figure 2: Some readers found the figure layout was a little difficult to follow at first and missed that Figure 2A is the quantitation for Figure 2A. Maybe consider revising layout for greater clarity.

o    Figure 3: We wondered whether the particle quantitation in different cellular compartments in Figure 3D would be more informative in the form of violin plots as elsewhere in the manuscript.

o    Figure 5A: Furthermore, readers thought that the capsid cartoon in Figure 5A could be improved to provide a stronger link to the data in Figures 5B and 5C. The description of the excess CA proteins that were not incorporated into the capsid proper was surprising to some readers who were less familiar with HIV literature. Could elements of this cartoon be incorporated into a revised Figure 1 describing the GCE/click-labelling procedure in greater detail?    

o    Figure 6: Minor point: white text on ET images is difficult to see.

MORE SUBJECTIVE CRITERIA (IMPACT)

1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2

●      A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.

o    The key finding can definitely be understood by a layperson, although more helpful cartoon figures would make the genetic code expansion strategy more clear. 

●      How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

o   This manuscript represents a considerable technical advance that opens up new possibilities to investigate capsid trafficking and uncoating and takes some initial steps towards applying the technology. The technology was not fully extended and used to address such questions in this paper.  

2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A

●  Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

●  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

o    N/A