Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly

Addressing Antiretroviral Drug Resistance with Host-Targeting Drugs—First Steps towards Developing a Host-Targeting HIV-1 Assembly Inhibitor

“…As Deng et al did not use such techniques to isolate assembly intermediates, it is likely that they were studying other complexes in addition to assembly intermediates, such as translating Gag associated with ribosomes, newly synthesized Gag, Gag undergoing degradation, and Gag in complexes that have partially dissociated. Indeed, the Gag-containing ribosomal complexes described by Deng et al [52] are almost certainly not assembly intermediates, since assembly intermediates contain proteins that are not found in ribosomes, such as the RNA granule proteins DDX6, AGO2, and DCP2, and lack the abundant small ribosomal protein S6 [29,41,46,49,50,54]. Notably, complexes containing Gag and RNA granule proteins are not biochemical artifacts since they have also been found in situ using two different imaging techniques in multiple cell types [29,41,46,49,50,54].…”

“…In a recent paper Deng et al argue against the existence of the stepwise pathway for HIV-1 assembly [52], but for multiple reasons this study is problematic. First, Deng et al conclude that all Gag-containing complexes that comigrate with ribosomes likely consist of Gag-binding to ribosomes based solely on their finding that recombinant Gag-GFP purified from bacteria can bind to purified ribosomes, presumably nonspecifically, and without showing that Gag binds to ribosomes in cells by a similar mechanism [52]. They did not address the possibility that complexes containing Gag bound to ribosomes, if they exist in cells, could co-exist with similar sized Gag-containing complexes of an entirely different composition such as assembly intermediates.…”

“…This technique is problematic since it is well known that Gag epitopes become inaccessible as Gag multimerizes [55]. Given that completed immature capsids contain thousands of Gag proteins [56], the detection of mainly Gag monomers and dimers by Deng et al (Figure 3 in [52]) raises the possibility that their approach simply excluded late assembly intermediates. Adding to that concern, no positive control was provided to demonstrate that their method is capable of capturing higher order Gag multimers.…”

“…Adding to that concern, no positive control was provided to demonstrate that their method is capable of capturing higher order Gag multimers. In a third problematic approach, Deng et al argue that the smaller Gag-containing complexes found in cells do not behave like assembly intermediates since they are labeled equivalently during SILAC pulse labeling [52]. However, given that assembly happens very rapidly (in <15 min based on imaging in [23,57]), a pulse label of 2 h, the shortest pulse used by Deng et al [52], would be expected to label many cohorts of assembling Gag and would actually approximate steady-state labeling which lacks the resolution needed to dissect the kinetics of a highly transient process.…”

“…In a third problematic approach, Deng et al argue that the smaller Gag-containing complexes found in cells do not behave like assembly intermediates since they are labeled equivalently during SILAC pulse labeling [52]. However, given that assembly happens very rapidly (in <15 min based on imaging in [23,57]), a pulse label of 2 h, the shortest pulse used by Deng et al [52], would be expected to label many cohorts of assembling Gag and would actually approximate steady-state labeling which lacks the resolution needed to dissect the kinetics of a highly transient process. In contrast, the pulse-chase conditions that originally identified the HIV-1 assembly intermediates involved a much shorter pulse of radiolabel (4-15 min) followed by unlabeled chase periods of varying lengths, allowing small cohorts of assembling Gag to be followed as the size of these Gag-containing complexes changed over time and thereby establishing the progression of a small population of labeled Gag through complexes of increasing size [37,39].…”