Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

Abstract

HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT.

Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate

“…Numerous reports demonstrate that NC plays roles in reverse transcription and integration (e.g., Buckman et al, 2003;Marchand et al, 2006;Poljak et al, 2003;Racine et al, 2016;Thomas et al, 2006). Reverse transcription also requires IN interaction (Tekeste et al, 2015).…”

The Life-Cycle of the HIV-1 Gag–RNA Complex

“…Cryo-electron tomography and subtomogram averaging highlighted the importance of the primary cleavage between p2 and NC domains of Gag to initiate dimer RNA stabilization, while the ensuing cleavages are necessary to complete the process [ 175 ]. This change in NC RNA chaperone activity during proteolytic processing [ 60 , 176 , 177 , 178 ] is likely to regulate reverse transcription through multiple mechanisms including the promotion of stable tRNA annealing [ 179 , 180 ], facilitation of strand transfer [ 181 , 182 , 183 ], regulation of reverse transcription initiation [ 184 , 185 , 186 ], and the general remodelling of RNA structure, possibly to prevent reverse transcriptase stalling [ 187 , 188 ]. Recently, the impact of the RNA genome on proteolytic processing has also started to be evaluated.…”

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

“…Several studies have highlighted the relationship between the biological role of NCp7 molecules and their density on RNA. The initial high number of NCp7 molecules on the viral RNA dimer enabling its full coating is thought to play an essential protective role inhibiting an early onset of reverse transcription 42 . Then, when the NCp7:nucleotide ratio decreases, the protein role likely switches from genome protection to reverse transcription chaperoning 6 .…”