Citation

  • Authors: Raghunath G. et al.
  • Year: 2022
  • Journal: Viruses 14 1636
  • Applications: in vitro / DNA / jetPRIME
  • Cell type: HEK-293T/17
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T/17, 293T/17

Method

The HIV-1 pseudoviruses were produced by transfecting HEK293T/17 cells with JetPRIME transfection reagent. To produce viruses for Laurdan staining, HEK293T/17 cells seeded in 10 cm dishes were transfected with 2.68 µg of HXB2 Env, 3.56 µg of pR9∆Env∆Nef, 1.2 µg of pcRev, 0.3 µg of mRFP-Vpr, 0.3 µg of either CMV-SERINC2-iHA, CMV-SERINC5-iHA, or empty pcDNA3.1 vector. To generate viruses for NR staining, the same plasmids were used except that 0.6 µg of GFP-Vpr was used instead of mRFP-Vpr. The HEK293T/17 cells and DNA transfection mix were incubated overnight at 37 ◦C in 5% CO2 after which time the transfection medium was replaced with high-glucose DMEM without phenol red with 10% FBS. To produce immature viruses, DMEM containing 300 nM Saquinavir was used to inhibit HIV-1 protease. Forty-eight hours post-transfection, the supernatants were collected, passed through 0.45 µm filters, concentrated with Lenti-X concentrator, aliquoted, and stored at −80 ◦C. The p24 content of viral preparations was determined by ELISA, as previously described

Abstract

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5's structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5's effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus' sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

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