Fusion of Mature HIV-1 Particles Leads to Complete Release of a Gag-GFP-Based Content Marker and Raises the Intraviral pH

Abstract

By imaging the release of a GFP-based viral content marker produced upon virus maturation, we have previously found that HIV-1 fuses with endosomes. In contrast, fusion at the cell surface did not progress beyond a lipid mixing stage (hemifusion). However, recent evidence suggesting that free GFP can be trapped within the mature HIV-1 capsid raises concerns that this content marker may not be released immediately after the formation of a fusion pore. To determine whether a significant portion of GFP is trapped in the mature capsid, we first permeabilized the viral membrane with saponin. The overwhelming majority of pseudoviruses fully released GFP while the remaining particles exhibited partial loss or no loss of content. The extent of GFP release correlated with HIV-1 maturation, implying that incomplete Gag processing, but not GFP entrapment by mature capsids, causes partial content release. Next, we designed a complementary assay for visualizing pore formation by monitoring the intraviral pH with an additional pH-sensitive fluorescent marker. The loss of GFP through saponin-mediated pores was associated with a concomitant increase in the intraviral pH due to equilibration with the pH of an external buffer. We next imaged single HIV-cell fusion and found that these events were manifested in a highly correlated loss of content and increase in the intraviral pH, as it equilibrated with the cytosolic pH. Fused or saponin-permeabilized pseudoviruses that partially lost GFP did not release the remaining content marker under conditions expected to promote the capsid dissociation. We were thus unable to detect significant entrapment of GFP by the mature HIV-1 capsid. Together, our results validate the use of the GFP-based content marker for imaging single virus fusion and inferring the sites of HIV-1 entry.

SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins

“…Fusion of HXB2pp containing or lacking SER5-HA or SER2-HA culminates in the release of mCherry through fusion pores larger than 4 nm in diameter (Fig. 3, A and B) (28). As expected, SER2-HA-containing particles and control virions fused equally well, whereas fusion of SER5-HA particles was markedly inhibited (Fig.…”

“…HXB2pp were co-labeled with YFP-Vpr and the viral content marker Gag-iCherry containing an "internal" mCherry tag (28). Fusion of HXB2pp containing or lacking SER5-HA or SER2-HA culminates in the release of mCherry through fusion pores larger than 4 nm in diameter (Fig.…”

“…HIV-1 pseudoviruses used in the BlaM assay and single virus experiments were produced by transfection of HEK293T/17 cells, as described previously (23,28). Cells were transfected with the following amounts of plasmids per 100-mm dish: 3 g of Env, 4 g of pR9⌬Env or pR9⌬Env⌬Nef, 1.5 g of pBJ5 or pBJ5-SER2-HA or pBJ5-SER5-HA (unless indicated otherwise), 2 g of BlaM-Vpr, and 0.5 g pcRev.…”

Early cytoplasmic uncoating is associated with infectivity of HIV-1

“…Multiple groups have shown that the generation of HIV-1 particles with a Gag-GFP polyprotein (HIV-iGFP) releases free GFP after proteolytic viral maturation. The liberated GFP then becomes an intravirion fluid phase marker (41)(42)(43). We previously demonstrated that a minority subset of the fluid phase GFP is trapped within the capsid structure (42,44).…”

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

“…Single-virus fusion assays were performed by imaging fluorescently labeled ASLVpp entry in CV-1 and A549 cells expressing the TVA950 or TVA800 receptor isoform, based on previous protocols (59,65). Briefly, cells were grown on collagen-coated number 1.5 glass-bottomed Petri dishes (MatTek, MA) up to 90% confluent in FluoroBrite DMEM with 10% FBS.…”