The Antiviral Restriction Factors IFITM1, 2 and 3 Do Not Inhibit Infection of Human Papillomavirus, Cytomegalovirus and Adenovirus

<div><p>Type I interferons (IFN-α and β) induce dynamic host defense mechanisms to inhibit viral infections. It has been recently recognized that the interferon-inducible transmembrane proteins (IFITM) 1, 2 and 3 can block entry of a broad spectrum of RNA viruses. However, no study to date has focused on the role of IFITM proteins in DNA virus restriction. Here, we demonstrate that IFN-α or -β treatment of keratinocytes substantially decreases human papillomavirus 16 (HPV16) infection while robustly inducing IFITM1, 2 and 3 expression. However, IFITM1, 2 and 3 overexpression did not inhibit HPV16 infection; rather, IFITM1 and IFITM3 modestly enhanced HPV16 infection in various cell types including primary keratinocytes. Moreover, IFITM1, 2 and 3 did not inhibit infection by two other DNA viruses, human cytomegalovirus (HCMV) and adenovirus type 5 (Ad5). Taken together, we reveal that the entry of several DNA viruses, including HPV, HCMV, and Ad5 is not affected by IFITM1, 2 and 3 expression. These results imply that HPV, and other DNA viruses, may bypass IFITM restriction during intracellular trafficking.</p></div

IFITM1 overexpression delays degradation of HPV16 L1 capsid protein in primary keratinocytes.

<p>HFKs were infected with 10,000 vge/cell of HPV16 virions and placed at 4°C for 1 h to inhibit endocytosis. Unbound virions were washed away and cells were incubated at 37°C for the indicated time points. Cells were trypsinized to remove non-internalized virions, and then lysed to detect internalized virions by western blotting for L1 capsid protein. Detection of β-actin was used as a loading control. Boxed bands are from a longer exposure. M, mock. *Non-specific band.</p

Overexpression of IFITM1 and 3 enhances HPV16 infection in epithelial cell lines and primary keratinocytes.

<p>HeLa (A & B), A549 (C & D), HaCaT (E & F), and HFK (G & H) cells expressing the indicated c-Myc-tagged IFITMs were inoculated with HPV16-LucF pseudovirions and incubated for 48 h. HPV16 infectivity was measured, as described in Fig. 1. Infectivity data was normalized to the vector alone control and shown as mean % infectivity from quadruplicate samples representative of at least two independent experiments with standard error. <i>P</i>-values were calculated as described in Fig. 1. Significant differences (*<i>p</i> <0.05, **<i>p</i> <0.01, ****<i>p</i> <0.0001) compared to vector transduced cells are marked with asterisks. IFITM protein expression in HeLa (B), A549 (D), HaCaT (F) and HFK (H) cells was measured by western blotting with an anti-c-Myc antibody. Detection of β-actin was used as a loading control. PageRuler plus ladder (Thermo) was used to approximate molecular weights: c-Myc-IFITM1, 18 kD; c-Myc-IFITM2 21 kD; c-Myc-IFITM3, 20 kD; β-actin, 46 kD.</p

Ad5 and HCMV infections are not affected by overexpression of IFITM1, 2 and 3.

<p>HeLa cells overexpressing IFITM1, IFITM2, or IFITM3 were infected with recombinant (A) Ad5-CMV-GFP or (B) HCMV TB40E mCherry and infectivity was measured by flow cytometry at 48 hpi. Gates were set based on the uninfected control and the percentage of fluorescent cells was quantified using FlowJo software (A & B). The results are presented from at least three independent experiments with standard error. (C) HeLa cells overexpressing IFITM proteins were infected with S-protein pseudotyped SARS-CoV luciferase viruses. Infectivity was measured by Bright-Glo Luciferase Assay System (Promega), normalized to the vector alone control, and shown as percent infectivity from triplicate samples representative of two independent experiments. <i>P</i>-values were calculated as described in Fig. 1. Significant differences (**<i>p</i> < 0.01) compared to vector-transduced cells are marked by asterisks.</p

Knockdown of endogenous IFITM1 or IFITM3 does not affect HPV16 infectivity.

<p>(A) Total RNA was isolated from HeLa cells stably expressing scrambled shRNA or shRNA targeting IFITM1 or IFITM3. Expression levels of IFITM1 and IFITM3 mRNA were measured by RT-qPCR and normalized by β-actin mRNA levels. Data are presented as percent change in mRNA expression of knockdown cells compared to cells with scrambled shRNA. (B) Cells in (A) were infected with HPV16-LucF and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096579#pone-0096579-g001" target="_blank">Figure 1</a>.</p

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div

Inhibition of NFκB activation induces autophagy in THP-1 cells.

<p>(<b>A</b>) Control THP-1 cells (0.1% DMSO) and THP-1 cells subjected to serum starvation, 5 µM BAY, <i>MTB</i> infection, or both <i>MTB</i>+BAY for 24 hrs, followed by immunoblotting of nuclear-free whole cell lysates for LC3 and β-actin. A representative immunoblot of three independent experiments is shown. (<b>B</b>) Human THP-1 cells were transduced with lentivirus-GFP-LC3 and differentiated into macrophages, followed by infection with <i>MTB</i> for 24 hrs in the absence or presence of 5 µM BAY. The cells were fixed and stained with DAPI to visualize the nuclei (blue) and the number of GFP-positive punctae were quantified. <i>Upper panel</i>, representative immunofluorescence images of three independent experiments; <i>lower panel</i>, average number of GFP-LC3 punctae per cell. The data shown represent the mean ± SEM of duplicate wells/condition from three independent experiments. (<b>C</b>) <i>MTB</i>-infected THP-1 cells treated with BAY were incubated with or without 3-MA, an inhibitor of the early phase of the autophagic pathway. After 48 hrs, the cells were lysed and nuclear-free whole cell lysates (20 µg per lane) were separated by SDS-PAGE and immunoblotted for LC3-I, LC3-II and β-actin. The bar graph represents the relative densities of the LC3-II bands normalized for their corresponding β-actin bands for two independent experiments. (<b>D</b>) THP-1 cells were infected with <i>MTB</i> H37Rv alone, <i>MTB</i>+5 µM BAY, or <i>MTB</i>+BAY+6 mM 3-MA for 4 days and cell-associated <i>MTB</i> was quantified. Data shown are mean ± SEM from two independent experiments performed in duplicates. *p<0.05, **p<0.01, ***p<0.001.</p

TNFα and IFNγ levels in MDM and AM infected with <i>MTB</i> H37Rv with or without NFκB inhibition.

<p>Primary human (<b>A</b>) MDM or (<b>B</b>) AM were infected with <i>MTB</i> H37Rv and after 1 hr, 24 hrs, 4 days, or 8 days of infection, supernatants were assayed for TNFα by ELISA and IFNγ by electrochemiluminescence. Data shown are estimated means with standard error bars from linear mixed model fits, based on seven independent experiments (MDM) or nine independent experiments (AM). *p<0.05 and **p<0.01 for cytokine expression in macrophages infected with <i>MTB</i> alone (closed circles) vs. <i>MTB</i>+BAY (closed squares). Control = (open circles) and BAY = (open squares).</p

Inhibition of NFκB activation by BAY 11-7082 reduces the viability of intracellular <i>MTB</i> in human macrophages.

<p>(A) THP-1 cells, (B) MDM, or (C) AM were pretreated with 0.1% (v/v) DMSO (open squares) or 5 µM BAY (closed circles) for 1 hr, followed by infection with <i>MTB</i> H37Rv. One hr, 4 days, or 8 days after infection, the cells were lysed and cultured for <i>MTB</i>. (D) Differentiated THP-1 cells were pretreated with 0.1% (v/v) DMSO (open squares) or 5 µM BAY (closed circles) for one 1 hr, followed by infection with <i>MTB</i>-H37Rv-GFP. One hr, 4 days, or 8 days after infection, fluorescent intensity was measured by Cytofluor II microplate fluorometer. Data shown as mean ± SEM. n = 4 for THP-1 cells in (A) and n = 2 for THP-1 cells in (D), n = 7 volunteers for MDM, n = 9 volunteers for AM. *p<0.05, **p<0.01, ***p<0.001.</p

Diagram of the mechanisms by which NFκB activation promotes the intracellular survival of <i>MTB</i>.

<p>Based on our experimental findings, NFκB activation enhanced the intracellular survival of <i>MTB</i> through inhibition of apoptosis and autophagy in infected macrophages. Since NFκB can also induce the production of its inhibiting molecule IκBα (blue line) and NFκB inhibition of autophagy could potentially prevent degradation of IKK (red line), the ultimate effect of NFκB on survival of intracellular <i>MTB</i> in macrophages is likely a complex process. IKK = IκBα kinase.</p