Introduction
Foamy viruses (FVs) belong to the
Spumaretrovirinae subfamily of
Retroviridae, and their replication strategy is intermediate between the
Hepadnaviridae and
Retroviridae. [
1,
2]. Envelope proteins are essential for FV replication and the budding of FVs is strictly envelope glycoprotein-dependent since their Gag proteins lack membrane-targeting signals and must interact with envelope proteins for membrane-targeting ability [
3,
4]. Similar to the hepatitis B virus S protein, FVs release Env-only subviral particles [
3]. FVs have a wide range of hosts and infect primates, felines, bovines, and equines [
5‐
8]; however, there have been few studies on the effects of cellular factors on FV replication.
The innate immune response plays an important role in antiviral therapy. Interferon (IFN)-induced transmembrane proteins (IFITMs) are activated by the interferon stimulating genes (ISGs), which have recently become an area of research focus on the antiviral immune response due to their broad-spectrum antiviral effects [
9]. At present, there are five genes encoding IFITM proteins in humans, including
IFITM1,
IFITM2,
IFITM3,
IFITM5, and
IFITM10 [
10]. IFITM1-3 proteins can be significantly upregulated by IFNs and are ubiquitously expressed in human tissues in the absence of IFN induction; however, IFITM5 and IFITM10 protein expression was not induced by IFN [
11]. IFITM1, IFITM2, and IFITM3 play a role in embryonic development, signal transduction, tumorigenesis, and antiviral activities [
12,
13]. IFITM5, which is involved in bone mineralization and maturation, is only expressed in osteoblasts, whereas the function of IFITM10 remains unclear [
14,
15]. Some other species have been reported to exhibit homologous genes in the IFITM family, suggesting that IFITMs have important and conserved functions [
16].
At present, the antiviral spectrum of IFITMs encompasses over 20 viruses from 12 families [
15]. These include DNA viruses, enveloped RNA viruses and non-enveloped RNA viruses [
17‐
19]. There are several viruses with severe pathogenicity in humans that are inhibited by IFITMs, including human immunodeficiency virus (HIV) [
20], Ebola virus (EBOV) [
21], influenza A virus (IAV) [
22,
23], Zika Virus (ZIKV) [
24] and severe acute respiratory syndrome coronavirus (SARS-CoV) [
25]. Among those with antiviral activity, IFITM3 is the best characterized [
26]. Moreover, there are some viruses that are not inhibited by IFITMs, including murine leukemia virus (MLV) [
27], adeno-associated virus (AAV) [
28], lymphocytic choroid plexus bacterial meningitis (LCMV) [
29], and arenavirus (LASV) [
15]. Further research is required to clarify the molecular mechanisms underlying IFITM antiviral activity and why some viruses can escape IFITM inhibition.
At present, the comprehensive antiviral mechanism of IFITM primarily includes IFITM-mediated inhibition of viral entry by inhibiting viral fusion to the plasma membranes and lysosomal or endosomal membranes, rather than relying on specific recognition of viral components to restrict virus entry [
9,
26]. IFITM proteins can also regulate the endosomal or lysosomal pH [
30]. The conformation of some viral envelope proteins (e.g., hemagglutinin) changes at a low endosomal pH, mediating hemifusion of viral and endosomal membranes [
31]. In addition, IFITM3 can inhibit the replication of some non-enveloped viruses by regulating the function of late endosomes [
18]. IFITMs have also been demonstrated to reduce the infectivity of some newly generated viruses. For example, IFITM proteins can be colocalized with HIV-1 Env and Gag and become part of the nascent generated virus particles, inhibiting virion entry into new host cells [
20]. Recently, IFITM proteins have been found to inhibit HIV-1 protein synthesis and thus limit viral infection [
32]. Prototype foamy virus (PFV) is a type of FV. However, whether the replication of PFV, an enveloped virus that entry target cells in a pH-dependent manner [
33], is affected by IFITMs remains unclear. In this study, we demonstrated that IFITM1-3 inhibits PFV replication by inhibiting PFV entry and reducing the number of PFV envelope protein.
Materials and methods
Plasmid constructs
Full-length infectious clone of PFV (pcPFV) was kindly provided by Maxine L. Linial (Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) [
34]. The pCMV-Tag2B-Tas plasmid was constructed by inserting the 9434 to 10,336 nucleotide region containing
orf1 into pCMV-Tag2B. HA-tagged IFITM1-3 eukaryotic expression plasmids were constructed by inserting the cDNA of human
IFITM1,
IFITM2, and
IFITM3 into pCMV-3HA vectors (Clontech, Mountain View, CA, USA). Insert the cDNA encoding human
IFITM3 into pQCXIP-Flag vector to construct a pQCXIP-Flag-IFITM3 plasmid. The PFV envelope encoding sequence was inserted into pCMV-3HA or pCE-puro-3×FLAG to construct the pCMV-3HA-Env or pCE-puro-3×Flag-Env plasmid. The pEGFP-C3 vector was purchased from BD Biosciences (Clontech, Mountain View, CA, USA). A pair of double-stranded oligonucleotides targeting
IFITM3 was inserted into the pSIREN-RetroQ vector (Clontech, Mountain View, CA, USA) to obtain the pSIREN-RetroQ-shIFITM3 plasmid.
Cell culture and transfection
Human embryonic kidney (HEK293T), PFVL (BHK21-derived indicator cell lines containing the
luciferase gene initiated by the PFV LTR) [
35], HT1080, and HeLa cells were incubated in Dulbecco's modified Eagle's medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco) at 37 °C in 5% CO2. Plasmid transfection was performed using polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) reagent according to the manufacturer's instructions [
36].
Virus production and infection
The pcPFV plasmid was transfected into HEK239T cells and the cell culture supernatant was collected 48 h after transfection and filtered through a 0.45 µm filter to obtain the PFV virus stock solution, which can be stored at 4℃. PFV viral titers were measured by infection of PFVL cells [
37]. Target cells were infected with the virus stock and fresh culture medium was replaced at 8 h post-infection. After 48 h of infection, 1/20 of the infected cells or 500 µL of cell culture supernatant were co-incubated with PFVL cells to test the infectivity of the virus produced by replication in target cells. The levels of PFV protein expression in the infected cells were analyzed by Western blotting.
Generation of knockdown cell lines
We used small hairpin RNA (shRNA) to screen IFITM3 knockdown cell lines. The target sequences of IFITM3 shRNA and control shRNA were 5′-TCCCACGTACTCCAACTTCCA-3′ and 5′-GAAGTAAGCGATATACATA-3′, respectively. HEK293T cells were co-transfected with 1 µg pSIREN-RetroQ-shIFITM3 or pSIREN-RetroQ-shControl plasmids, 1 µg pMVL-Gag/Pol, and 0.5 µg pVSV-G. HT1080 cells were infected with the cell culture supernatants at 48 h after transfection. At 48 h post-infection, knockdown HT1080 cells were selected by medium containing puromycin (2 μg/mL). The following primer sequences were used to detect IFITMs mRNA levels in HT1080-shIFITM3 cells by qPCR: IFITM1-up: 5′-ATCCTGTTACTGGTATTCGG-3′; IFITM1-low: 5′-TATAAACTGCTGTATCTAGG-3′; IFITM2-up: 5′-GTTGGTCGTCCAGGCCCAGC-3′; IFITM2-low: 5′-CTGTGGGGACAGGGCGAGGA-3′; IFITM3-up: 5′-GCTGATCTTCCAGGCCTATG-3′; IFITM3-low: 5′-GATACAGGACTCGGCTCCGG-3′.
Luciferase reporter assay
Following a co-incubation with cell culture supernatants (containing virus particles) or PFV-infected cells, luciferase levels of PFVL cells were determined using a luciferase reporter assay system (Promega, Madison, WI, USA). The corresponding results were the average of three independent experiments.
Alu-PCR
HT1080 cells transfected with IFITM3 expression plasmid or control vector were infected with the PFV virus stock, and the total DNA of infected cells was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Duesseldorf, Germany) 30 h post-infection. Cells treated with 10 µM of the integrase inhibitor raltegravir or 10 µM of the reverse transcriptase inhibitor AZT were used as controls, the degree of PFV genome integration was detected by semi-quantitative PCR and real-time PCR using Alu-PCR primers.
The extracted total DNA (100 ng) was used as template, and 10 µM of Alu1, Alu2 and SpA primers were added for PCR. PCR conditions were 95 °C for 5 min for 1 cycle; 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 3 min, for 22 cycles; 68 °C for 7 min for 1 cycle. Next 2 µL PCR products were used as the template, and 10 µM Lambda and Nested-R primers were added for the second PCR. PCR conditions were: 95 °C for 5 min for 1 cycle; 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 30 s, for 22 cycles; 68 °C for 7 min for 1 cycle. Using the extracted total DNA as a template, the gapdh gene was amplified as a control. PCR conditions were 95 °C for 5 min for 1 cycle, 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s, for 22 cycles, and 68 °C for 7 min for 1 cycle. The following primer sequences were used: SpA: 5′-ATGCCACGTAAGCGAAACTTAGTATAATCATTTCCGCTTTCG-3′; GAPDH-up: 5′-AACAGCGACACCCACTCCTC-3′, GAPDH-low: 5′-CATACCAGGAAATGAGCTTGACAA-3′; Lambda: 5′-ATGCCACGTAAGCGAAACT-3′; NestedR: 5′-GAAACTAGGGAAAACTAGG-3′; Alu1: 5′-TCCCAGCTACTGGGGAGGCTGAGG-3′, Alu2: 5′-GCCTCCCAAAGTGCTGGGATTACAG-3′.
Western blotting
The cell lysates were placed on ice for 30 min after adding lysis buffer (1% NP-40, 3% Glycerol, 2 mM EDTA, 50 mM Tris, 150 mM NaCl) to the cell samples to be tested. The proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Cincinnati, OH, USA). The PVDF membrane was blocked with 5% nonfat milk for 45 min, after which the PVDF membrane was incubated with primary antibodies for 1.5 h and incubated with peroxidase-conjugated secondary antibodies for 45 min. Immunoreactive protein signals were detected by chemiluminescence (Merck Millipore, Darmstadt, Germany).
Antibodies
The following antibodies were used in the protein detection analysis in this study: polyclonal rabbit anti-IFITM3 (1:2000; cat. no. 11714-1-AP, Proteintech, Chicago, IL, USA), monoclonal mouse anti-HA (1:3000; cat. no. H3663, Sigma-Aldrich, St. Louis, MO, USA), monoclonal mouse anti-GFP (1:2000; cat. no. sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse anti-Tubulin (1:5000; cat. no. sc-32293, Santa Cruz), monoclonal mouse anti-GAPDH (1:5000; cat. no. sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; cat. no. sc-2005, Santa Cruz), and HRP-conjugated goat anti-rabbit IgG (1:5000; cat. no. sc-2004, Santa Cruz), and monoclonal mouse anti-Flag (1:5000; cat. no. F1804, Sigma-Aldrich) antibodies. We use purified Tas or PFV Gag (180–433 aa) proteins as immunogens to immunize BALB/c mice to obtain antibodies against the corresponding proteins. Since part of the Bet protein overlaps with the 88 amino acids at the N terminus of the Tas protein, the Bet protein can be detected using anti-serum prepared with the Tas protein as an immunogen.
Virus entry assay
HT1080 cells were infected with a PFV stock solution and incubated at 4℃ for 1 h to ensure virus attachment to the cell membrane surface but not entry into cells. The cells were washed with PBS to remove any unattached virus, and the cells were cultured at 37℃ after replacing the culture medium. After 0 h, 2 h, 4 h 6 h and 8 h, the cells were harvested to extract the total RNA from the infected cells. The number of viral genomes in the cells was detected by RT-qPCR to indicate the level of viral entry. The amount of viral genome was indicated by the level of Gag gene expression. The following primers were used for RT-qPCR: Gag forward (5′-AATAGCGGGCGGGGACGACA-3′), Gag reverse (5′-ATTGCCACGCACCCCAGAGC-3′); GAPDH forward (5′-AACAGCGACACCCACTCCTC-3′), GAPDH reverse (5′-CATACCAGGAAATGAGCTTGACAA-3′).
Statistical analysis
The data were presented as the mean ± standard deviations (SD) of the results of three independent experiments. The data were analyzed using GraphPad Prism Version 8.0 (GraphPad software Inc., San Diego, CA, USA). When the P value was greater than 0.05, the difference was not significant (ns). A P value less than 0.05 indicated a statistically significant difference (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Discussion
At present, IFITMs have been demonstrated to antagonize a variety of viruses, including several serious pathogenic viruses, including human immunodeficiency virus (HIV) [
43], Ebola virus (EBOV) [
21], and Zika Virus (ZIKV) [
24]. In addition, there are some viruses that are able to escape the confinement of IFITMs, such as murine leukemia virus (MLV) [
27], adeno-associated virus (AAV) [
28] and arenavirus (LASV) [
15]; however, the antiviral properties of IFITMs and the reason that some viruses are able to evade inhibition by IFITMs remain unclear. Further research on the effect of IFITMs on diverse viral replication will help clarify the antiviral mechanism of IFITMs.
In this study, we demonstrated that IFITM1-3 could significantly inhibit PFV, and interestingly they inhibited PFV to a similar degree. Previous studies have shown that different IFITMs tend to inhibit the same virus to different degrees. For example, compared with IFITM1 and IFITM2, IFITM3 has a significantly strong effect on influenza A virus (IAV) [
23] and IFITM3 can inhibit Zika virus (ZIKV) replication more effectively than IFITM1 [
24]. Moreover, the degree of HIV-1 inhibition varies among IFITM members (IFITM3 > IFITM2 > IFITM1) [
20], and is related to many factors. Such factors include physiological and biochemical characteristics of IFITMs (e.g., the differences in IFITM localization) and the molecular characteristics of virus replication (e.g., the position of the virus entering the target cell). A similar degree of PFV-mediated inhibition by IFITM1-3 may be related to the unique replication strategy of PFV, the specific mechanism of which requires further study.
IFITM1, IFITM2 and IFITM3 have been reported to inhibit syncytia formation and cell–cell fusion induced by a several classes viral fusion proteins, and the degree of inhibition depends on the cell type [
44]. In this study, we reported that IFITM3 also inhibited syncytium formation induced by PFV infection in HT1080 cells. IFITMs can inhibit the entry of a variety of viruses, most of which are viruses that enter cells in a pH-dependent manner. As a virus that enters cells in a pH-dependent manner [
33], IFITM3 also inhibits PFV entry into host cells. Studies have shown that IFITM1 is primarily localized at the plasma membrane, whereas IFITM2 and IFITM3 are more localized to intracellular compartments and co-localize with lysosomal-associated membrane protein 1 (LAMP1), Rab7, or CD63 [
9,
45]. Moreover, PFV Env-mediated fusion occurs at both the plasma membrane and in endosomes [
46]. Therefore, although IFITM3 is more likely to inhibit PFV fusion in the endosomal membrane, the specific mechanism requires further exploration. Considering that IFITM1, IFITM2, and IFITM3 have a strong inhibitory effect on PFV replication, further investigation is warranted regarding whether IFITM1 can inhibit PFV fusion in the plasma membrane and whether IFITM2 inhibits PFV fusion in endosomes.
For FVs, envelope glycoprotein mediates viral entry into host cells and are also essential for viral budding [
3]. The IFITM proteins were reported to inhibit the late step of feline foamy virus (FFV) replication without effect on early entry step, but the exact mechanism remains unclear [
41]. In this study, we also found that IFITM3 downregulated the number of PFV envelope protein, and this reduction was associated with IFITM3 promoting envelope protein degradation via the lysosomes. These results suggest that, unlike FFV, IFITM3 inhibits both PFV entry into target cells and late step in the PFV life cycle. Previous studies have shown that IFITM3 can impair the processing of the HIV-1 envelope protein and degrades the envelope protein through lysosomes [
42,
47]; however, the Ebola glycoprotein is insensitive to IFITM3 [
42], suggesting that not all viral glycoproteins inhibited by IFITMs are affected by IFITMs.
Overall, these results suggest that in addition to inhibiting PFV entry, IFITM3 also reduces the abundance of envelope proteins that are essential for viral replication, and PFV can be inhibited via these two mechanisms.
Acknowledgements
We thank Maxine L. Linial (Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) for sharing the valuable plasmids. We also thank Chen Liang (McGill University, Montreal, Quebec, Canada) for helpful comments and discussion of the paper and for providing the valuable plasmids.
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