The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity - PubMed (original) (raw)

The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity

Rui Jia et al. PLoS One. 2015.

Abstract

The interferon-inducible transmembrane (IFITM) proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1) strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1NL4-3) is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117-125), which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117-125) mutant diminishes HIV-1NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117-125) to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117-125), mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.

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Conflict of interest statement

Competing Interests: CL is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.

Figures

Fig 1. Effect of IFITM1 on HIV-1 replication.

(A) SupT1 cells were stably transduced to express IFITM1 under the induction by doxycycline. HIV-1 strains BH10, NL4–3 and NLENY1-IRES (an infectious derivative of NL4–3) were used to infect SupT1 cells with or without doxycycline induction. Virus production was monitored by measuring levels of viral reverse transcriptase (RT) activity in culture supernatants at different time intervals. The infection experiments were performed three times and the result of one representative experiment is shown. Control SupT1 cells were transduced with the empty retroviral vector pRetroX-Tight-Pur. (B) Illustrated is the amino acid sequence of IFITM1. The two predicted transmembrane domains are highlighted. The positions of three deletion mutations are indicated. Both the wild type and mutated IFITM1 have a Flag tag attached to the N-terminus. Levels of the wild type and mutated IFITM1 in stably transduced SupT1 cells were examined by western blotting. An amount of 500 μg/ml doxycycline was used to induce IFITM1 expression. Levels of tubulin were probed as internal controls. (C) Replication of NL4–3 and BH10 in SupT1 cells stably expressing IFITM1 mutants Δ(117–125), Δ(112–125) or Δ(108–125). Virus production was determined by measuring levels of viral RT activity in culture supernatants. A representative result of three independent infections is shown.

Fig 2. Effects of wild type IFITM1 and its mutants on HIV-1 entry.

(A) BlaM-Vpr containing HIV-1NL4–3 particles were used to infect SupT1 cells that express wild type IFITM1 or its mutants Δ(117–125), Δ(112–125) or Δ(108–125). Levels of virus entry were determined by monitoring the cleavage of CCF2 by BlaM-Vpr. Similar entry experiments were performed with HIV-1NL4–3 that was pseudotyped with VSV G protein. (B) Summary of the results from three independent virus entry experiments.

Fig 3. Subcellular localization of wild type IFITM1 and its mutants Δ(117–125), Δ(112–125) or Δ(108–125).

(A) HEK293 cells were transfected with plasmid DNA expressing wild type IFITM1 and its mutants Δ(117–125), Δ(112–125) or Δ(108–125) together with Rab5-GFP. Localization of IFITM1 and its mutants were determined by immunostaining with anti-Flag antibody. Nuclei were stained with DAPI. Images shown represent the major subcellular distribution of each protein. The periphery of the immunostained cells was outlined with white dot lines on the basis of the cell morphology observed in phase contrast channel. The fluorescence intensity for IFITM1 and its mutants (in red) at the cell periphery and cell interior was determined using the ImageJ software. The percentage of IFITM1 at the cell periphery was then calculated, and the results obtained from at least 10 cells were shown as mean±standard derivation. (B) HEK293 cells were transfected with plasmid DNA expressing the wild type IFITM1 or its variant IFITM1-KDEL that has the ER retention signal attached to the C-terminus. Cells were either treated with dynasore (160 μM) or DMSO as control. IFITM1 and IFITM1-KDEL were detected by immunostaining with anti-Flag antibody. The endogenous calreticulin was stained with anti-calreticulin antibody. Nuclei were stained with DAPI. Images shown represent the major subcellular distribution of each protein.

Fig 4. Effect of mouse IFITM1 on HIV-1 infection.

(A) Alignment of IFITM1 sequences from different species. Human IFITM1 and mouse IFITM1 are highlighted in red letters. The two predicted transmembrane domains are indicated. (B) Replication of HIV-1NL4–3 in SupT1 cells that express mouse IFITM1 (mIFITM1) or human IFITM1 (hIFITM1) under induction by doxycycline. Control cells are transduced with empty retroviral vector pRetroX-Tight-Pur. Growth of viruses was monitored by measuring levels of viral RT activity in culture supernatants at various time intervals. One representative data is shown. (C, D) Effect of mouse IFITM1 on virus entry that was mediated by HIV-1NL4–3 Env or VSV G protein. Virus entry is determined by monitoring the cleavage of CCF2. Results of three independent experiments are summarized in the bar graph.

Fig 5. Identification of HIV-1 protein conferring resistance to IFITM1.

(A) Illustration of chimeric viruses that were generated by exchanging DNA fragments between HIV-1BH10 and HIV-1NL4–3. The recognition sites by ApaI, SalI and BamH1 in HIV-1 DNA are shown. AS, between ApaI and SalI sites; SB, between SalI and BamHI sites; Senv, between SalI site and the beginning of Env; envB, between the Env initiation site and BamHI site. (B) Replication of chimeric viruses BH(AS), BH(SB), NL(AS) and NL(SB) in SupT1 cells expressing IFITM1 under induction by doxycycline. (C) Replication of BH10, NL4–3 and their chimeric viruses in IFITM1-expressing SupT1 cells.

Fig 6. Effect of IFITM1 on HIV-1BH10 and HIV-1NL4–3 in the one-round infection assay.

SupT1 cells were treated with doxycycline (500 μg/ml) for 16 hours to induce IFITM1 expression before infection with HIV-1BH10 (A, B) or HIV-1NL4–3 (C, D). Forty hours after infection, cells were harvested and stained with FITC-conjugated anti-p24 antibody. Infected cells were scored by flow cytometry (shown in A and C). Amounts of viruses made in the supernatants were determined by ELISA to measure HIV-1 p24/CA (shown in C and D). Results of three independent experiments are summarized in the bar graph.

Fig 7. Cell-to-cell transmission of BH10, NL4–3 and their chimeric viruses.

(A) Cell-to-cell transmission between SupT1 cells that did not express IFITM1. The donor SupT1 cells were infected with HIV-1, then mixed with target cells that were labeled with cell tracker BMQC. Infected cells were monitored by immunostaining viral p24 using FITC-conjugated anti-p24 antibody. The infected target cells are scored in the Q2 window of the flow cytometry plots. Relative transmission efficiency was calculated by dividing the percentage of infected target cells (Q2/(Q1+Q2)) with that of infected donor cells (Q3/(Q3+Q4)). Results of three independent transmission experiments are summarized in the bar graph. (B) Cell-to-cell transmission from control SupT1 cells that do not express IFITM1 to SupT1 cells that express exogenous human IFITM1. The control SupT1 cells were first infected with HIV-1 and used as the donor cells to transmit viruses to target cells. Transmission efficiency was calculated as described above. *** denotes p value <0.01.

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The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity - PubMed (original) (raw)