Reviewer 1's Review

“This manuscript presents new data on an important topic. The experiments are almost flawless from a technical point of view and, for the most part, appropriate conclusions are drawn. I have some comments for the purpose of improving the manuscript. Some might be addressed in a new version of the manuscript; some might be simply discussed with the editor. Points numbered 1 to 3 below are the most critical ones; the rest of them are merely for text enhancement.

1. There is a lack of references concerning the nature of 2-LTR junctions, especially since this is central to this work. For instance, in the introduction, where the authors state that "this sequence is similar... to the palindromic consensus found at integration sites" without a citation. Later in the same paragraph, they write that "The palindromic sequence in the 2-LTR circles... is a general feature of retroviruses" yet they cite only one paper which is about one virus (HIV-1). References are also needed in figure 1A, which shows the 2-LTR junction for HIV-1 and other retroviruses.

2. To what extent does the sequence used to construct the PalA/PalB ODNs represent actual 2-LTR junctions? Do all HIV-1 strains have this exact sequence at their 2-LTR junction? In infected cells, what is the % of 2-LTR junctions that are a perfect match to the consensus?

3. The results shown here are somewhat surprising, as IN does not seem to cut oligos representing the weakly palindromic integration sites (which it must cut in vivo), while it does cut LTR:LTR junctions (though 2-LTR circles are considered replication dead-ends). Regarding the fact that WP1 and WP2 were not internally cut by HIV-1 IN: I noticed that the expected target site in these ODNs was quite different from the consensus integration site as published by Holman and Coffin. Thus, it is not clear whether the authors actually consider these ODNs to be representative of consensus integration sites? (The authors use the words "mimic the weak palindrome consensus" in the second paragraph of the result section, again without any reference).”

N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.