Reevaluation of the requirement for TIP47 in human immunodeficiency virus type 1 envelope glycoprotein incorporation

Abstract

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.

Fig 1

Interaction of purified TIP47 with its natural ligands in SPR assays. (A) Sensorgrams depicting the interaction of the cytoplasmic tail of CD-M6PR with sensor chip-immobilized human TIP47. Results for GST-tagged CD-M6PR cytoplasmic tail protein at concentrations of 0.134, 0.269, 0.538, 1.075, and 2.15 μM are shown. (B) Sensorgrams depicting the interaction of the Rab9 GTPase with sensor chip-immobilized human TIP47. Results for His-tagged Rab9(1–177) proteins at concentrations of 8.6, 17.3, 34.6, 134.3, and 276.6 nM are shown. Black lines indicate experimental data, whereas red lines indicate fitting to a 1:1 Langmuir binding model with a parameter included for mass transport. Kinetic values are as follows: for CD-M6PR-TIP47, k_a = (1.3 ± 0.2) × 10⁴ M⁻¹ · s⁻¹; k_d = 4.5 × 10⁻² · s⁻¹; K_D = 3.6 μM; for Rab9-TIP47, k_a = (4.2 ± 2.0) × 10⁴ M⁻¹ · s⁻¹; k_d = (1.0 ± 0.1) × 10⁻³ · s⁻¹; K_D = 24.4 nM.

Fig 2

The N terminus of TIP47 is required for its interaction with HIV-1 MA in SPR assays. HIV-1 MA at a concentration of 250 nM was passed over full-length TIP47 and Δ1–186 TIP47, and the responses were recorded. (A) Representative sensorgrams for these interactions; (B) mean responses of three experiments in graphical form. Error bars represent 1 standard deviation.

Fig 3

MA interacts with TIP47 in NMR-based assays. ¹H-¹⁵N HSQC NMR spectra obtained upon titration of ¹⁵N-labeled HIV-1 myrMA (50 μM) (A) and myr(−)MA (50 μM) (B) with intact TIP47; [TIP47]:[MA] = 0:1 (black), 0.5:1 (green), and 1:1 (red).

Fig 4

The N terminus of TIP47 is required for its interaction with TIP47 in NMR-based assays. (A, B) ¹H-¹⁵N HSQC NMR spectra obtained for ¹⁵N-labeled HIV-1 myrMA (50 μM) (A) and myr(−)MA (50 μM) (B) upon titration with Δ1–112 TIP47; [Δ1–112 TIP47]:[MA] = 0:1 (black), 1:1 (red). (C) ¹H-¹⁵N HSQC NMR spectra obtained for ¹⁵N-labeled Δ1–112 TIP47 upon titration with myrMA; [MA]:[Δ1–112 TIP47] = 0:1 (black), 2:1 (green), and 4:1 (red).

Fig 5

Depletion of TIP47 in HeLa cells does not impair HIV-1 Env incorporation, virus release, or infectivity in HeLa cells. Cells were transfected with 30 pmol nontargeting siRNA (NT) or a pool of four TIP47-specific siRNAs. (A) Transfected cell lysates were probed with antibodies specific for tubulin and TIP47 (left panel); the ratio of TIP47 to tubulin was quantified by using a FluorChem SP Imaging System (Alpha Innotech) (right panel) (means ± SD; n = 3). (B) Control and TIP47-depleted cells were transfected with pNL4-3 and metabolically radiolabeled with [³⁵S]Cys; cell and virus lysates were immunoprecipitated with HIV-Ig. The relative ratios of virus-associated gp120 to p24(CA) and virus release efficiencies were quantified by phosphorimager analysis (means ± SD; n = 3). (C) Virus-containing supernatants were harvested from control and TIP47-depleted cells, normalized for RT activity, and used to infect the TZM-bl indicator cell line. Graphs show average levels of infectivity with a virus input of 50,000 RT cpm. Luciferase activity was measured 2 days postinfection (means ± SD, n = 3).

Fig 6

Overexpression of TIP47 in HeLa cells does not enhance HIV-1 Env incorporation or virion infectivity. HeLa cells were cotransfected with pNL4-3 and either GFP [(−)Ctrl] or TIP47 expression vectors (pCMV-TIP47) at the indicated DNA concentrations. (A) Transfected cell lysates were probed with antibodies specific for tubulin and TIP47. (B) Cells were metabolically radiolabeled with [³⁵S]Cys; cell and virus lysates were immunoprecipitated with HIV-Ig. The relative ratios of virus-associated gp120 to p24(CA) were quantified by phosphorimager analysis (± SEM; n = 3). Using a paired t test, both samples overexpressing TIP47 were considered not significantly different from the negative control (two-tailed P values > 0.3) (C) Virus-containing supernatants were harvested from control and TIP47-overexpressing cells, normalized for RT activity, and used to infect the TZM-bl indicator cell line with four different virus inputs. Graphs show average levels of infectivity with a virus input of 50,000 RT cpm. Luciferase activity was measured 2 days postinfection (± SEM; n = 3).

Fig 7

Depletion of TIP47 in Jurkat cells does not disrupt HIV-1 Env incorporation, infectivity, or virus release. (A) TIP47 depletion in Jurkat cells using shRNAs. TIP47 and tubulin were detected by Western blotting of puromycin-resistant Jurkat cells after transduction with TIP47-targeted shRNAs or nontargeting (NT) shRNA. Average protein levels relative to tubulin are shown. Error bars indicate SEM (n = 3). (B) HIV-1 Env incorporation efficiency in TIP47-depleted Jurkat cells. Cells were metabolically radiolabeled with [³⁵S]Cys; cell and virus lysates were immunoprecipitated with HIV-Ig (left panel). Average levels of Env incorporation are shown (right panel). Error bars indicate SEM (n = 4). Using a paired t test, none of the results were considered significantly different from the NT control results (P > 0.05). (C) HIV-1 release efficiency in TIP47-depleted Jurkat cells. Average virus release efficiency normalized to NT shRNA-transduced cells is shown. Error bars indicate SEM (n = 4). Using a paired t test, none of the results were considered significantly different from the NT control results (P > 0.01). (D) Infectivity of HIV-1 produced from TIP47-depleted Jurkat cells. Infectivity was detected by luciferase activity in infected TZM-bl cells, and results were averaged. Infectivity assays were performed with four different virus inputs, normalized for RT activity. Graphs show average levels of infectivity with a virus input of 25,000 RT cpm. Error bars indicate SEM (n = 4). Using a paired t test, the increases in infectivity were marginally significant relative to the controls (P = 0.1 for shRNA 2 compared to mock sample, and P = 0.02 for shRNA 2 compared to NT sample).

Fig 8

TIP47 depletion does not significantly affect HIV-1 replication in the Jurkat T-cell line. Jurkat cells transduced with shRNA 1 or 2 targeting TIP47 (#1 or #2) or nontargeting shRNA (NT) or Jurkat cells with no shRNA (mock) were transfected with the HIV-1 molecular clone pNL4-3 and passaged by dilution with fresh medium every 2 to 3 days. Culture supernatants were collected at each passage, and virus replication was monitored by RT activity.