Peer review of: Structural basis of host protein hijacking in human T-cell leukemia virus integration (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is an exciting and important manuscript describing the characterization of the HTLV-1 IN:DNA 
intasome, in complex with the B56γ(B´γ) subunit of an essential host enzyme, protein phosphatase 
2A (PP2A), using cryo-electron microscopy. The structure is novel and shows the interactions of two 
PP2A B56γ molecules in complex with an IN tetramer in the presence of a branched DNA molecule. 
This interaction is primarily through a loop region localized between the IN CCD and CTD domains, 
which serves different functions in related retroviruses. Mutagenesis studies and viral titers validate 
the importance of this inter-domain region. 

Specific comments: 

1. The manuscript states that the B56γ was “essential for a stable HTLV IN-DNA complex formation”. 
Comment whether any intasome complex in the absence of B56γ was observed on any of the size and 
MW analysis presented in the manuscript (Extended Fig 2). 

2. Results, Extended Data Fig. 2. This is an essential figure to analyze the stoichiometry of the 
complex. 2A. Molecular standards should be presented in a plot to clearly indicate their exact 
molecular weights. Consistency between the fraction numbers of Figure 2A, and 2B should be very 
convincing. 

3. Page 7, first paragraph. Comment on whether the presence of B56γ functions on stability of the 
intasome complex alone or has an effect on target-site preference. 

4. Page 14 and extended Fig. 6. The stimulation by B56γ to IN integration is reported as “modest”. 
Was this quantified and how does it compare to previous studies cited (ref. 28) 

5. Extended Fig 3. Provide addition information regarding the Sso7D domain. Is it structured? Does it 
sterically block assembly of potential higher ordered oligomers or B56γ binding? 

6. Method of HTLV intasome preparation. After dialyses of intasome assembly mixture against buffer 
with 100 mM NaCl, the assembly mixture is supplemented with 150 mM NaCl, and incubate for 30 
min. Please clarify the purpose of this additional step. 



Reviewer #2 (Remarks to the Author): 

It was previously shown that serine/threonine protein phosphatase PP2A, including fragment B56y11-
380, interacts with delta-retroviral INs and specifically stimulates HTLV concerted integration 
(Maertens GN et al. 2016). However, it remained unclear which regions of IN were involved in this 
interaction and how PP2A-B56y stimulates HTLV integration. In this work, Bhatt et al. attempt to 
addresses these questions with the cryo-EM structure of the HTLV1 strand transfer complex intasome 
bound to the human protein B56y11-380, as well as biochemical and cell biology experiments. This 
work provides an important contribution to the field as it shows the first structure of the HTLV 
intasome and its interaction with the host protein B56y11-380. These can aid drug development. In 
addition, this is the first structural report for a Deltaretroviral intasome, which contributes to a better 
understanding of intasome structural variability. Despite the importance of these findings, the major 
issue in the manuscript is the confusing conclusions from the biochemical and cell biology 
experiments. In the absence of further clarification (and some control experiments), the conclusions in 
the current report are not fully justified. 
Major points 

The authors state that B56y is essential for the formation of a stable intasome complex, but the in 
vitro integration assay (Extended Fig. 6) shows that HTLV1 intasomes are apparently equally active in 
the presence or absence of B56y. This is somewhat confusing. If B56y is essential to form stable 
intasomes, why does its addition have a negligible effect on integration in vitro? What, if anything, is 
B56y or PP2A doing in vitro? 

Following the same rationale, the cellular data of PP2A / B56y in vivo is generally confusing. Fig. 3 
shows an infectivity assay with multiple constructs supporting the role of PP2A-B56y as a specific 
enhancer for HTLV1 infection. Knockdown of B56y can be interpreted as either (1) affecting viral 
infectivity or (2) affecting normal cellular function, and thus, indirectly, infectivity. One cannot 
currently distinguish between these two possibilities. Notably, there is no control to reintroduce B56y 
back and rescue infectivity/function. This is a key experiment. Similarly, the overexpression data is 
confusing. If mutant B56g (R197A, or the double mutant L194A/R197A) cannot bind to IN, why would 
overexpressing it compromise infectivity? Wouldn’t one expect overexpression of the mutant to have a 
minimal effect? Overexpression mutants could also be affecting the host. The overall effect, in cells, 
appears to behave more like a dominant-negative, affecting the host more than it actually affects the 
virus. 

The authors should clarify how the shRNA-mediated knockdown was conducted. Was the region 
spanning residues 11-380 on B56y targeted? 

It seems to me that the authors were able to obtain a very nice structure, and then wanted to boost 
the importance of the structural findings with more mechanistic data. But the latter is complicated and 
confusing, leading to more questions than answers. What is PP2A doing and what is its relevance in 
HTLV integration? Is it playing an active role in integration? Or is it simply passively hijacked by IN, 
perhaps in a way that might be similar to HTLV Tax protein. 

Minor points 

Co-factor typically refers to small molecules. Better to use “accessory factor” to describe PP2A 

Shouldn’t Extended Data Figure 2 come before Extended Data Figure 1? 

Extended Data Figure 5 should have the FL HTLV-1 IN as a positive control. Presumably, it will behave 
similarly to HTLV-1 CCD-CTD. 

Extended Fig. 3 is confusing. I would suggest authors to provide a global view of the intasome and 
zoom in panels from boxed regions, so that readers can visualize the context of the HTLV intasome 
with respect to each panels (Ext. Fig. 3c-h). 

Local resolution coloring (Fig. 1E-F) can go into Extended Data. The authors also should show a map-
to-model FSC to determine the quality of the model fit, and preferably (given that there’s fairly strong 
preferred orientation), a measure of directional resolution (e.g. 3D FSC) 

Increase font size for numbers comprising the X and Y axes of plots in Ext. Fig. 2a and Ext. Fig. 5. 

Does Sso7d impact oligomerization? 

Are the NTD/CTD domains of the outer IN visible 

The authors mention on page 4 that HTLV1 resembles RSV intasomes due to the same target site 
duplication length comprising 6 bp, although the intasome organization resembles the tetrameric PFV 
intasome. Since this is the first report of a Deltavirus intasome, it would be useful to have an 
extended figure showing global differences in protomer organization between intasomes from HTLV-1, 
PFV, and RSV.