Evidence that the endosomal sorting complex required for transport-II (ESCRT-II) is required for efficient human immunodeficiency virus-1 (HIV-1) production.
BACKGROUND
Egress of a number of different virus species from infected cells depends on proteins of the endosomal sorting complexes required for transport (ESCRT) pathway. HIV has also hijacked this system to bud viruses outward from the cell surface. How ESCRT-I activates ESCRT-III in this process remains unclear with conflicting published evidence for the requirement of ESCRT-II which fulfils this role in other systems. We investigated the role of ESCRT-II using knockdown mediated by siRNA and shRNA, mutants which prevent ESCRT-I/ESCRT-II interaction and a CRISPR/Cas9 EAP45 knockout cell line.


RESULTS
Depletion or elimination of ESCRT-II components from an HIV infected cell produces two distinct effects. The overall production of HIV-1 Gag is reduced leading to a diminished amount of intracellular virion protein. In addition depletion of ESCRT-II produces an effect similar to that seen when ESCRT-I and -III components are depleted, that of a delayed Gag p26 to p24 +p2 cleavage associated with a reduction in export of virion particles and a visible reduction in budding efficiency in virus producing cells. Mutants that interfere with ESCRT-I interacting with ESCRT-II similarly reduce virus export. The export defect is independent of the decrease in overall Gag production. Using a mutant virus which cannot use the ALIX mediated export pathway exacerbates the decrease in virus export seen when ESCRT-II is depleted. ESCRT-II knockdown does not lead to complete elimination of virus release suggesting that the late domain role of ESCRT-II is required for optimal efficiency of viral budding but that there are additional pathways that the virus can employ to facilitate this.


CONCLUSIONS
ESCRT-II contributes to efficient HIV virion production and export by more than one pathway; both by a transcriptional or post transcriptional mechanism and also by facilitating efficient virus export from the cell through interactions with other ESCRT components.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. bm432@cam.ac.uk.
Bo
B
Meng
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. natasha.ip@cantab.net.
Natasha C Y
NC
Ip
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. l.j.p21@hotmail.com.
Liam J
LJ
Prestwood
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. g.abbink@vumc.nl.|Centre for Childhood White Matter Disorders, VU University Medical Centre, Amsterdam, The Netherlands. g.abbink@vumc.nl.
Truus E M
TE
Abbink
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. amll1@medschl.cam.ac.uk.
Andrew M L
AM
Lever
D001692:Biological Transport; D064113:CRISPR-Cas Systems / Q000235:genetics; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D014771:Virion / Q000378:metabolism; D057074:Virus Release; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D054301:gag Gene Products, Human Immunodeficiency Virus