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

Sergi Padilla-Parra,Mariana Marin,Nivriti Gahlaut,Rolf Suter,Gregory B Melikyan,Naoyuki Kondo

doi:10.1371/journal.pone.0071002

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.

Highlights

The HIV-1 envelope glycoprotein (Env) glycoprotein initiates viral fusion with the host cell membrane through sequentially engaging CD4 and coreceptors, CXCR4 or CCR5 [1,2]
Single virus fusion has been imaged using particles co-labeled with a GFP-based viral content marker and a lipophilic dye incorporated into the viral membrane
As suggested in [27], the majority or all iGFP molecules produced by proteolytic cleavage of HIV-1 Gag-iGFP is trapped within the mature ‘‘sealed’’ capsid, this content marker would not be fully released through a fusion pore (Fig. 1B)

Summary

Introduction

The HIV-1 Env glycoprotein initiates viral fusion with the host cell membrane through sequentially engaging CD4 and coreceptors, CXCR4 or CCR5 [1,2]. Kinetic measurements of virus-cell fusion revealed that HIV-1 acquires resistance to a membrane-impermeant peptide fusion inhibitor (targeting surface-accessible viruses) much earlier than to low temperature, which blocked all fusion events. This result suggests that HIV-1 escapes from inhibitory peptides, such as enfuvirtide [23], by entering an endocytic pathway and fusing with endosomes at a later time. The fact that the viral content release was not associated with loss of a lipid marker demonstrated that full fusion occurred in small intracellular compartments that were not connected to the plasma membrane. It appears that HIV-1 fusion with the plasma membrane is arrested at a hemifusion stage upstream of fusion pore formation

Methods

Results

Conclusion