Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

Abstract

Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P2). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

“…Endosomes close to the viral factory also lacked the dilated appearance of endosomes filled with cholesterol. The reduction in ASFV infectivity due to U1866A is comparable to the impact of inhibiting endosomal acidification with bafilomycin A (BAF) (15,18).…”

“…Immunofluorescence experiments were performed as previously described (18). Vero cells were fixed in PBS-4% paraformaldehyde (PFA) for 15 min and permeabilized with PBS-0.1% Triton X-100 or saponin (Sigma) for 10 min.…”

“…At 6 hpi, cells were washed with PBS and harvested by trypsinization. Flow cytometry experiments were performed by detection of infected cells with an anti-p30 monoclonal antibody as described elsewhere (18) or by GFP expression at 16 hpi when using recombinant viruses; 10 4 cells per tube in triplicates were scored and analyzed in a FACSCalibur flow cytometer (BD Sciences) to determine the percentage of infected cells under these conditions. The obtained infection rates were normalized to the corresponding controls.…”

“…Actin-dependent macropinocytosis also assists ASFV entry into cells (17). Once the virus is inside the endosome, viral decapsidation occurs in the acid pH of LE within a few minutes after entry (18). After uncoating, the virus needs to exit the endosome and gain access to the cytoplasm to start replication in a specialized structure called the viral factory (19).…”

Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

“…4C). This finding might reflect the maturation state of the endosome at the time the virus is delivered, since the pH gradually drops during endosomal maturation (47,48).…”