Pooled Rnai Screen Identifies Ubiquitin Ligase Itch As

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Pooled RNAi screen identiﬁes ubiquitin ligase Itch as crucial for inﬂuenza A virus release from the endosome during virus entry

Wen-Chi Sua,b,c, Yung-Chia Chena, Chung-Hsin Tsengd,e, Paul Wei-Che Hsua, Kuo-Feng Tungf, King-Song Jenga,f, and Michael M. C. Laia,b,c,d,e,1

aInstitute of Molecular Biology and fNational RNAi Core Facility, Academia Sinica, Taipei 11529, Taiwan; bResearch Center for Emerging Viruses, China Medical University Hospital, Taichung 40402, Taiwan; cChina Medical University, Taichung 40402, Taiwan; dDepartment of Microbiology and Immunology, College of Medicine and eCenter of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan 70101, Taiwan

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved September 20, 2013 (received for review June 29, 2013) Influenza viruses, like other viruses, rely on host factors to support therapies. Recently, several genome-wide siRNA-arrayed screening their life cycle as viral proteins usually “hijack,” or collaborate studies have been reported (3–7) that have identified several host with, cellular proteins to execute their functions. Identification factors, such as SON, CLK1 (6), and IFITM proteins (3), involved and understanding of these factors can increase the knowledge of in various steps of the viral life cycle. However, very few common molecular mechanisms manipulated by the viruses and facilitate host factors have been identified among these screens, indicating development of antiviral drugs. To this end, we developed a unique the intrinsic limitation of these screening methods (8). Neverthe- genome-wide pooled shRNA screen to search for cellular factors less, from bioinformatics analysis of these identified host genes important for influenza A virus (IAV) replication. We identified an some common pathways, such as the endocytic pathway, have been E3 ubiquitin ligase, Itch, as an essential factor for an early step in identified as essential for influenza virus infection. the viral life cycle. In Itch knockdown cells, the incorporation of In this study, we conducted a genome-wide pooled shRNA viral ribonucleoprotein complex into endosomes was normal, but screen and identified several factors in addition to the previously its subsequent release from endosomes and transport to the nu- identified factors. Among the factors, a ubiquitin ligase, Itch, cleus was retarded. In addition, upon virus infection, Itch was phos- was found to be a crucial component in the viral entry–uncoating phorylated and recruited to the endosomes, where virus particles process in the early stage of the viral life cycle. ICTH has been were located. Furthermore, Itch interacted with viral M1 protein described as having a role in proteolytic ubiquitination processes fi and ubiquitinated M1 protein. Collectively, our ndings unravel of various biological functions, including immune responses, a critical role of Itch in mediating IAV release from the endosome DNA repair, and cellular differentiation (9–11), as well as in and offer insights into the mechanism for IAV uncoating during nonproteolytic ubiquitination processes such as membrane protein fi virus entry. These ndings also highlight the feasibility of pooled sorting and cellular translocation (12, 13). In this study, we showed RNAi screening for exploring the cellular cofactors of lytic viruses. that Itch is crucial for IAV entry, acting in the release of the en- tering virion from the endosomal compartments. This protein thus nfluenza virus is an important human pathogen causing sig- fills the missing link in understanding the viral entry process. Inificant morbidity and mortality every year. Influenza A viruses (IAVs), belonging to the Orthomyxoviridae family, are enveloped Results RNA viruses with eight segments of single-stranded, negative- Implementation of a Genome-Wide RNAi Screen. To identify po- sense RNA encoding 11 viral proteins (1). Three viral proteins, tential cellular factors indispensable for viral replication, we namely, the surface glycoproteins hemagglutinin (HA), neur- developed a unique genome-wide RNAi screen. The human aminidase (NA) and the M2 ion channel protein, and the host shRNA library from the RNAi consortium (TRC) was used to cellular membrane-derived lipid bilayer build the viral envelope. transduce human A549 lung carcinoma cells. Notably, no more Underneath the envelope is the matrix protein (M1), a major structural protein. The core of the virus particle is the viral ri- Significance bonucleoprotein (vRNP), composed of viral RNA (vRNA) associated with the nucleoprotein (NP) and three components of Seasonal epidemics and recurring pandemics of influenza viru- the viral RNA polymerase complex (PA, PB1, and PB2). ses threaten public health and the global economy severely. At the beginning of infection, the HA protein binds to sialic Host factors that are essential for viral growth provide potential acid-containing receptors on the host-cell surface and elicits drug targets and are crucial for understanding the mechanism endocytosis of the viral particle, including its transportation of viral infection. This paper presents a unique genetic screen- through early and late endosomes. The viral envelope then fuses ing approach to identify such host factors. A cellular enzyme with the late endosome to go through viral uncoating, namely, termed Itch ubiquitin ligase was identified and found to be the release of vRNP from endosomes to the cytosol, and its essential for influenza viral entry into cells. It allows the viral subsequent import to the nucleus (2). Replication and tran- genome to escape from the trapping of the cells to initiate in- scription of vRNAs are carried out by the viral RNA-dependent fection. This host factor fills the critical gap in our understanding RNA polymerase (RdRp) and NP in the nucleus. In the late of the beginning event of influenza viral infection. stage of the viral life cycle, vRNP, M1, and viral envelope pro-

teins assemble the virion particles, which are then released from Author contributions: W.-C.S., K.-S.J., and M.M.C.L. designed research; W.-C.S., Y.-C.C., the cell surface to produce viral progeny. and C.-H.T. performed research; K.-S.J. contributed new reagents/analytic tools; W.-C.S., The high mutation rate of influenza virus leads to generation P.W.-C.H., and K.-F.T. analyzed data; and W.-C.S. and M.M.C.L. wrote the paper. of viral escape mutants, crippling the efficacy of vaccines and The authors declare no conflict of interest. antiviral agents targeting specificinfluenza viral proteins. An This article is a PNAS Direct Submission. alternative strategy that targets host factors, which possess ge- 1To whom correspondence should be addressed. E-mail: [email protected]. fl netic stability and are essential for in uenza virus replication, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. would thus be important for the development of new influenza 1073/pnas.1312374110/-/DCSupplemental.

17516–17521 | PNAS | October 22, 2013 | vol. 110 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1312374110 Downloaded by guest on September 28, 2021 than one shRNA was introduced to each cell; consequently, only (MOI) of 0.5. At 6 h p.i., the cell extract or total RNA was one gene was presumably silenced in any given cell. As illustrated prepared and used for further assays. Compared with the shLacZ in Fig. 1A, the shRNA-harboring cells were first established and control, which was transduced with an shRNA targeting a non- subsequently infected with influenza A/WSN/33 (WSN) virus. human gene, both Western blot and quantitative RT-PCR results Because IAV infection causes an extensive cytopathic effect, we showed that Itch silencing with shRNA clone 2 or 3 efficiently aimed to select surviving shRNA-containing cells, which signify reduced Itch levels and correspondingly decreased IAV repli- efficient inhibition of IAV replication by the particular trans- cation, as indicated by reduction of viral NP levels (Fig. 2 A and duced RNAi. As predicted, after several days mock shRNA- B). Moreover, transduction of wobble Itch mutants significantly transduced cells or naïve untransduced A549 cells were com- overcame the shRNA-induced Itch suppression and partially pletely dead whereas some surviving cells could be found in compensated the KD effect of Itch shRNA on NP expression, shRNA-transduced cells. The genomic DNA, which contains verifying the specificity of the effects of Itch shRNA clones 2 and shRNAs, was isolated from the pooled surviving cells and am- 3 (Fig. 2C). Next, we examined the Itch-silencing effect on virion plified for deep sequencing and identification of the encoded production. The progeny virus titer was reduced up to 10-fold in shRNA. The shRNA-targeted genes were categorized according Itch KD cells (Fig. 2D), further confirming that Itch is crucial for to the number of hit shRNA clones (Table S1). To reduce the the IAV life cycle. Additionally, silencing Itch also reduced the numbers of shRNAs acting through off-target effects, we defined replication of another IAV strain, A/Taiwan/3446/2002 (H3N2) genes to be “hits” if at least two unique shRNAs targeted the (Fig. 2E), suggesting that the requirement for Itch may be con- same gene. On the basis of this criterion, 110 genes (shown in served across IAV subtypes. Table S2), consisting of group I and group II, were selected as primary hits. Notably, compared with other RNAi screens, 26 Itch Is Involved in the Early Stage of the IAV Life Cycle. Itch has been genes out of 110 candidate genes had been previously identified revealed to modulate immunity through ubiquitination of its as important factors of influenza virus replication (8), such as substrates, including c-Jun and mitochondrial antiviral-signaling Rab5A (3, 4) and ATP6V1B2 (5, 6), strengthening the reliability protein (9, 15, 16). To clarify whether Itch affected IAV repli- of our screen method. The hit was further selected as a candidate cation via modulation of immunity, we examined the induction if it could show expression in EST analysis or in microarray of IFN response in Itch KD cells using a luciferase reporter β analysis (Fig. 1B). To focus on the cellular factors involved in the plasmid under the control of IFN- promoter and found that fl early stage of the viral life cycle, we carried out an imaging-based silencing Itch did not in uence IFN response compared with the A screen by staining the viral NP at 6 h postinfection (p.i.). The shLacZ control (Fig. S2 ). Thus, the effects of Itch KD on IAV images were analyzed to calculate the percentage of NP-positive replication were most likely due to direct effects of Itch on IAV fl cells. In total, 38 genes (shown in Table S2), hit by two unique replication. To delineate how Itch in uences IAV replication, we shRNAs showing at least 40% reduction of viral replication, were investigated the effect of silencing Itch at different viral life selected for assessing their functions in the viral life cycle. By using stages. We have shown that KD of Itch led to reduction of NP A B DAVID 6.7 software (http://david.abcc.ncifcrf.gov/home.jsp)to levels at 6 h p.i. (Fig. 2 and ), when most of the viral RNA classify gene functions, we obtained several enriched gene cate- replication and translation are taking place. Accordingly, we gories associated with various molecular functions such as proton- applied a minireplicon reporter assay to examine whether Itch transporting ATPase activity, cytoplasmic vesicle, and ubiquitin- affects RdRp activity. As expected, KD of nuclear RNA export dependent protein catabolic process (Fig. S1). factor 1, which regulates the nuclear export of viral mRNA (4, 17), inhibited RdRp activity; in contrast, KD of Itch had no ef- Identification of Itch As an Essential Cofactor for IAV Replication. fect on RdRp activity, suggesting that Itch may not play a role in B Previously, we have demonstrated that ubiquitination partici- viral RNA transcription and replication (Fig. S2 ). Next, we pated in regulating IAV RNA replication (14). Therefore, we assessed whether Itch is involved in the internalization of IAV by focused on studying the roles of ubiquitin-related genes in the detecting internalized viral matrix protein (M1) (18). For this IAV life cycle. The E3 ubiquitin ligase Itch, one of our identified purpose, A549 cells were incubated with IAV; the attached but candidates, was chosen for follow-up experiments. To verify that not internalized virion was removed by an acidic washing step · Itch is involved in IAV replication, Itch knockdown (KD) cells (PBS HCl, pH 1.3) before harvesting of cellular lysates (18). As C were infected with IAV (WSN virus) at multiplicity of infection shown in Fig. S2 , the internalized virus particles as represented by M1 proteins in the Itch KD cells were equivalent to those in the control cells. Thus, Itch most likely functions at a step after internalization of virus particles but before replication and A pooled shRNA B Procedure Gene number transcription of the genome. Primary screen using > 16,000 pool selection A549 cells Itch Participates in the Release of IAV from Endosomes. Next, we studied the fate of the internalized virus by examining the cel- Puromycin selection of shRNA Identifying hits lular distribution of IAV after virus entry. We observed that in positive cells (2 shRNAs) 110 Expansion of cell population Itch KD cells proportionally more vRNA was in the cytoplasm Selection of candidates than was in control cells, whereas the reverse was true in the Influenza A virus by gene expression nucleus (Fig. 3A), implying that the transport of viral RNA into (M.O.I. =1) 100 the nucleus in the early stage of the viral life cycle is defective in Secondary screen using HCS Cells with shRNA sub- Cells with control non- the Itch KD cells. To better track the subcellular distribution of pool shRNA encoding 38 vRNPs, we performed immunostaining of NP because NP is lentivirus or without Characterization of candidates lentivirus infction associated with viral genomic RNA. We found that 8% of NP- using bioinformatics analysis Analyze the staining cells showed nuclear staining of NP in the Itch KD cells shRNAs in these flu Validation of candidates at 60 min after infection, whereas 27% of the control cells showed resistant cells such staining (Fig. 3B and Fig. S3A), suggesting that silencing Itch Fig. 1. Genome-wide RNAi screen identifies host genes required for IAV inhibited nuclear import of IAV. replication. (A) Schematic presentation of pooled RNAi screen. (B) Outline of We next performed subcellular fractionation biochemically. Im-

the screening procedures. Numbers of genes associated with the process are munoblotting with nucleolin (for nucleus), Rab7 (for late endo- MICROBIOLOGY shown on the right side. HCS, high-content screening. some), and actin (for cytosol) showed satisfactory fractionation

Su et al. PNAS | October 22, 2013 | vol. 110 | no. 43 | 17517 Downloaded by guest on September 28, 2021 both control and Itch KD cells M1 could be detected in the peri- AB120 ITCH vRNA_NP nuclear region, apparently colocalized with late endosomes, im- ITCH shRNAs 100 plying that silencing Itch did not affect the entry of IAV into shRNA shLacZ #1 #2 #3 #4 #5 80 endosomes (Fig. S3C). At 60 min p.i., part of M1 could be NP 60 detected in the nucleus in the control cells; in contrast, very

ITCH 40 little M1 appeared in the nucleus in the Itch KD cells (Fig.

Relative RNA (%) Relative RNA 3D). In conclusion, our studies showed that KD of Itch re- Actin 20 tarded the release of virus from the endosomes. 0 Itch Colocalizes with IAV in the Endosomes. Having demonstrated that Itch might affect a step related to endocytosis of IAV, we C shRNA ITCH ITCH next examined whether Itch was associated with the endosome A549 cells Naive (wobble#2) Naive (wobble#3) upon IAV infection. The Flag-tagged Itch was overexpressed to facilitate observation. The Itch signal was diffuse in the cytoplasm in the uninfected cells; however, it became punctate, particularly ITCH ITCH in the perinuclear region in the IAV-infected cells, as early as 0.5 h p.i. (Fig. 4A). When the cells were treated with 0.05% saponin NP NP before ﬁxation of cells to remove most of the cytosolic Itch (20), the Itch signal became undetectable in the uninfected cells Actin Actin whereas the punctate stain of Itch was retained in the infected NP/Actin 1 0.19 1.3 0.51 NP/Actin 1 0.40 1.04 0.65 cells (Fig. 4B), suggesting that Itch was compartmentalized. To determine the nature of the compartments, we performed IAV_H3N2 D 14 E 12 10 8 AB 6 NP 200 35 180

Viral titer 4 30 160

(*10^5 pfu/mL) (*10^5 ITCH 2 140 25 0 120 20 Actin shLacZ shITCH#2 shITCH#3 100 80 cytoplasmic 15 shRNA 60 vRNA 10 40 nuclear vRNA -staining cells Relative vRNA Relative vRNA (%)

NP 5 20

Fig. 2. Silencing Itch reduces IAV replication. (A and B) The shRNA-trans- of Nucleic localization 0 0 duced A549 cells were infected with WSN at an MOI of 0.5. At 6 h p.i., the shLacZ shITCH#2 shITCH#3 shLacZ shITCH cells were harvested. (A) Immunoblotting against anti-NP and anti-Itch shRNA shRNA antibodies. Actin was used as loading control. (B) Cellular RNA was extracted C 10 min p.i. 60 min p.i. 120 120 and measured by quantitative RT-PCR. The levels of viral RNA and Itch mRNA 100 100 ± were normalized by GAPDH mRNA. Values represent the mean SD of three 80 80 cytosol cytosol independent experiments. (C) A549 cells were transduced with the lentivi- 60 60 endosome endosome ruses carrying the Itch wobble mutants to establish stable cell lines and then 40 40 nucleus nucleus 20 transduced with shLacZ (as control) or Itch shRNA clone 2 or clone 3 virus as distribution (%) 20 percent of vRNA distribution (%) percent of vRNA percent of vRNA indicated. After selection with puromycin, the shRNA-transduced naïve or 0 0 stable cells were reseeded and infected with WSN at an MOI of 0.5. Cells were harvested at 6 h p.i. and subjected to Western blot analysis with the indicated antibodies. The band intensities of NP and actin were quantiﬁed, shRNA shRNA and the relative NP/actin ratios are shown below the blots. (D) The shRNA- transduced A549 cells were infected with WSN at an MOI of 0.01. The D shLacZ shITCH supernatants were collected at 24 h p.i. and used for determining viral titer M1 Rab7 DAPI M1 Rab7 DAPI by plaque-forming unit assay in MDCK cells. (E) The experiment was performed the same way as in A except that H3N2 virus was used in place of WSN virus.

(Fig. S3B). To better evaluate the Itch KD effect on subcellular Fig. 3. IAV is confined in endosomes in Itch KD cells. (A) The shRNA-trans- distribution of vRNA, we included an additional treatment with duced A549 cells were infected with WSN strain (MOI 5) for 20 min. The bafilomycin A1 (BafA1), which is an inhibitor of IAV entry and cytoplasmic and nuclear viral RNA was extracted and used for quantitative + fusion via inhibition of vacuolar-type H ATPase (19). At 10 min RT-PCR. Each vRNA level was compared with that of the control, shLacZ. ± p.i., around 75% of total vRNA was in the endosome in the control Values represent the mean SD of three independent experiments. (B)The as well as Itch KD and BafA1-treated cells (Fig. 3C), whereas shRNA-transduced A549 cells were infected with WSN strain (MOI 5) for 30 min and fixed for immunofluorescence staining with anti-NP antibody and around 20% of vRNA was in the nucleus. At 60 min p.i., the vRNA DAPI. Values are shown as numbers of cells displaying nuclear localization of in the endosomal fraction dropped to 50% in the control cells; NP among 100 NP-staining cells. Values represent the mean ± SD of three correspondingly, the vRNA in the nuclear fraction increased to independent experiments. (C) The shRNA-transduced A549 cells and BafA1 40% (Fig. 3C). In contrast, the subcellular distribution of vRNA in (40 nM)-treated cells were infected with WSN (MOI 5) for 10 min or 60 min the Itch KD cells, similar to that in the BafA1-treated cells, was and then processed for subcellular fractionation. The fractions from different almost unchanged between 10 min p.i. and 60 min p.i. It is im- compartments were used for RNA isolation and determination of their vRNA portant to note that the amount of vRNA in the cytosol from Itch levels by quantitative RT-PCR. The percentage of vRNA was the ratio of the KD cells did not increase from 10 min p.i. to 60 min p.i., suggesting vRNA from each compartment to the vRNA from whole cells. Values represent the mean of two independent experiments. (D) The shRNA-transduced that silencing Itch retards the release of vRNA from endosome to A549 cells were infected with WSN (MOI 5) for 60 min, permeabilized with cytosol and its subsequent transport to the nucleus, instead of di- 0.05% saponin for 2 min, and then fixed for immunofluorescence stain with rectly blocking nuclear import of IAV from the cytosol. These anti-M1 (green) antibody, anti-Rab7 (red) antibody, and DAPI (blue).The results were confirmed by tracing M1 distribution. At 10 min p.i., in white arrowheads indicate the nuclear distribution of M1.

17518 | www.pnas.org/cgi/doi/10.1073/pnas.1312374110 Su et al. Downloaded by guest on September 28, 2021 fusion and release of vRNP from the endosomes (Fig. 6C). AB ﬁ 0.05% Saponin 0.05% Saponin Similarly, the M2 inhibitor, amantadine, which prevents acidi - Infection Infection cation of the virion, failed to inhibit phosphorylation of Itch (Fig. S5). Combining all of the results, we suggest that Itch is activated by endocytosis and recruited to endosomes, where it is involved in ubiquitination of M1 protein and the release of vRNP from endosomes to complete the viral entry process.

Fig. 4. Itch localizes in endosomes upon IAV infection. (A and B) Flag-tag- Discussion ged Itch-expressing A549 cells were infected or uninfected with WSN at an Many laboratories have used various strategies to search for the MOI of 50 for 30 min. After virus infection, the cells were processed without host factors involved in viral replication (3–7, 25). Although the (A) or with (B) 0.05% saponin treatment for 2 min, then ﬁxed and stained number of genes common among the various screens is low, with anti-Flag antibody (green) and DAPI (blue). probably owing to differences in screening approaches (8), the

costaining of Flag-tagged Itch and various endosomal markers. As shown in Fig. 5A, Itch partially colocalized with early endo- AB somal antigen (EEA1), a marker of early endosomes, and CD63, a marker of late endosomes/lysosomes. In addition, early in infection (0.5 h p.i.) Itch was also partially colocalized with IAV proteins HA and M1 (Fig. 5B). These results indicate that Itch and the IAV proteins are recruited into endosomes upon IAV infection. To determine whether there is an interaction between ITCH/EEA1/DAPI ITCH/CD63/DAPI ITCH/HA/DAPI ITCH/M1/DAPI Itch and IAV proteins, we performed a coimmunoprecipita- C Flag ITCH E HA- tagged M1 tion assay. Among the IAV structural proteins, M1 specifically HA-tagged GST HA NA M1 M2 Myc-Ub (KD) ITCH KD #2 #3 C 170 coprecipitated with Flag-tagged Itch (Fig. 5 ). To rule out 130 (KD) fi 95 170 possible nonspeci c binding to M1, we used another Flag- 72 130 95 55 tagged protein, Flag-tagged c-Jun. As shown in Fig. S4,M1 72 43 HA 55 coprecipitated with Itch but not c-Jun, strengthening the speci- 34 myc 43 ficity of the interaction between Itch and M1. Because Itch is an 26 Input (1/20) E3 ubiquitin ligase, we next examined whether its ligase activity is Input (1/20) 34 17 crucial for IAV replication. In the presence of a ligase-deficient 26

Itch mutant, C830A (21), the IAV replication was diminished, 17 Flag implying that ubiquitin ligase activity is important to IAV repli- HA (KD) cation (Fig. 5D). Having shown the association between M1 and 170 (KD) 130 170 Itch, we then assessed whether M1 could be ubiquitinated. In- 95 130 ) 72 95 55 72

terestingly, M1 could be ubiquitinated into multiple ubiquitinated ) species (Fig. 5E). Although high-stringency wash buffer was used 43 55

agarose HA 34 43 26 HA HA in this experiment to prevent M1 from coprecipitating with other agarose myc proteins nonspeciﬁcally, we still could not exclude the possibility 34

IP ( 17 that high-molecular-weight bands may represent M1-interacting 26 IP ( HA

proteins. Signiﬁcantly, the amounts of mono- and oligo-ubiquiti- Flag 17

nated M1 species were reduced in the Itch KD cells, suggesting HA that M1 is a substrate of Itch E3 ubiquitin ligase (Fig. 5E). Taken D C830A WT together, our results suggested that upon IAV infection Itch is NP recruited together with viral particles into the endosomes, where ITCH Itch may ubiquitinate M1 and facilitate release of viral RNP from the endosomes. Actin

Itch Is Phosphorylated at an Early Stage of the Viral Life Cycle. It has NP/Actin 1 0.54 1.3 been demonstrated that the ubiquitin ligase activity of Itch is Fig. 5. Itch interacts with M1 and mediates ubiquitination of M1. (A and B) activated by phosphorylation (22). We found that Itch began to Flag-tagged Itch-expressing A549 cells were infected with WSN at an MOI of be phosphorylated at 10 min p.i.; the phosphorylation level was 50 for 30 min then treated with 0.05% saponin and processed for immu- highest at 30 min p.i. and then gradually declined at 45 min and nofluorescence staining with the following antibodies: (A) anti-Flag anti- 60 min p.i. (Fig. 6A). The Itch protein level remained constant body (green), anti-EEA1 or anti-CD63 antibody (red), and DAPI (blue) or (B) anti-Flag antibody (green), anti-HA or anti-M1 antibody (red), and DAPI during the same period. This observation supported the roles of (blue). (C) HEK293T cells were cotransfected with Flag-tagged Itch and the Itch in the endocytosis of IAV during virus entry. To further indicated HA-tagged plasmids. The cell lysates were prepared and used for correlate the activated Itch with endocytosis, we used inhibitors immunoprecipitation with HA agarose. The cell lysates or immunoprecipi- of endosomal trafficking, including LY294003 and wortmannin, tates were resolved on SDS/PAGE and immunoblotted with anti-HA or anti- which are inhibitors of early endosomal fusion via inhibition of Flag antibody. (D) Naïve, WT Itch-overexpressing and C830A Itch mutant- overexpressing A549 cells were infected with WSN at an MOI of 0.5. At 6 h PI3-kinase (23), and BafA1 and NH4Cl, which are known to block acidification as well as HA-induced fusion with the late p.i. the cells were harvested for Western blot analysis against anti-NP and endosomal membrane (24). As shown in Fig. 6B, the presence of anti-Itch antibodies. The relative NP/actin ratios are shown below the blots. wortmannin and LY294002 reduced the phosphorylation of Itch (E) The shRNA-transduced HEK293T cells were cotransfected with HA-tagged ∼ M1 and Myc-tagged ubiquitin. The lysates were prepared and used for im- by 45% at 30 min p.i., implying that Itch phosphorylation is munoprecipitation with HA agarose. The cell lysates or immunoprecipitates linked to the execution of endocytosis. Intriguingly, the BafA1 were resolved on SDS/PAGE and immunoblotted with anti-Myc or anti-HA

and NH4Cl treatment did not affect phosphorylation of Itch, sug- antibody. shLacZ- transduced HEK293T cells were used as control cells. The MICROBIOLOGY gesting that Itch is activated before the virus–endosomal membrane arrows indicated the mono- and oligo-ubiquitinated M1 species.

Su et al. PNAS | October 22, 2013 | vol. 110 | no. 43 | 17519 Downloaded by guest on September 28, 2021 A IAV (min) 0 10 30 45 60 functions after the viral fusion step. Nevertheless, this conclusion P-ITCH will require support from other experimental approaches. Here, we propose a possible mechanism of Itch function in ITCH viral entry, as shown in Fig. 7. IAVs first bind to sialic acid- Actin containing receptors at the cell surface, internalize, and consequently elicit endocytosis. Because phosphorylation of Itch was B DMSO Wortmannin LY294002 inhibited by wortmannin and LY294002 but not by BafA1 nor +IAV (min) 0 30 60 0 30 60 0 30 60 NH4Cl (Fig. 6 B and C), Itch is likely phosphorylated after early P-ITCH endosomal trafficking and thereafter resides in late endosomes, as supported by our observation that Itch became punctate and ITCH compartmentalized (Fig. 4 and Fig. 5A). Actually, this phe- Actin nomenon is similar to previous findings that with distinct stimuli P-ITCH/Actin (%) 28 100 54 14 55 43 28 60 21 Itch becomes punctate (20) or localizes to the endosomal system (12). It has been recognized that before viral fusion with the late fl C DMSO BafA1 NH4Cl endosomal membrane in uenza M2 ion channel protein actively pumps protons from the endosome into the interior of the virion, +IAV (min) 0 30 60 0 30 60 0 30 60 lowering the pH inside the virion (31), resulting in dissociation of P-ITCH M1 from vRNP, thereby facilitating the dissociation of vRNAs ITCH from M1 (32, 33). Our findings of the involvement of the ubiquitin ligase Itch and ubiquitination of matrix protein M1 may Actin explain how the virion becomes unstabilized, thus facilitating the P-ITCH/Actin (%) 35 100 90 61 140 90 66 117 94 release of RNP. We speculate that acidification can induce Fig. 6. Itch is phosphorylated in the early stage of the IAV life cycle. (A) conformational alteration of the viral envelope and destabilize A549 cells were incubated with WSN (MOI 5) at 4 °C for 1 h and refreshed the membrane, thereby facilitating Itch’s entry into the virus with warm medium at time 0. The cell extracts were harvested at the in- particle and access to M1. Subsequently, ubiquitination of M1 dicated time points and analyzed by Western blotting with anti–phospho- causes conformational change or degradation of M1, thereby Itch or anti-Itch antibody. Actin was used as loading control. (B and C) A549 triggering vRNP egress into the cytosol, and eventually transport cells were treated with the inhibitors for 1 h and then infected with WSN to the nucleus. Overall, our studies advanced the mechanistic (MOI 5) at 4 °C for 1 h in the presence of inhibitors. After virus infection, the understanding of IAV replication, in particular the viral un- cells were refreshed with warm medium and harvested at 30 min or 60 min p.i. The cell extracts were analyzed by Western blotting with anti–phospho-Itch coating process, and are applicable to other screens of interest. or anti-Itch antibody. The band intensities of P-Itch and actin were quantified, and the relative P-Itch/actin ratios are shown below the blots. The concen- Materials and Methods trations of inhibitors are as follows: wortmannin, 1 μM; LY294002, 50 μM; Cell Lines, Plasmids, Reagents, and Viruses. Human lung adenocarcinoma – BafA1, 40 nM; and NH4Cl, 50 mM. epithelial cells (A549), Madin Darby canine kidney cells (MDCK), and human embryonic kidney cells (HEK293T) were used for this study. The A/WSN/33 (H1N1) (WSN) strain of IAV was mainly used in the studies. Detailed in- genes revealed by different screenings display meaningful net- formation on cell culture, plasmids, reagents, and lentivirus preparation is works of host–influenza virus interactions (8). To develop more given in SI Materials and Methods. effective methods to discover cellular factors required for viral shRNA Screen. The genome-wide TRC shRNA library, consisting of 81,925 replication, we established a pooled RNAi screen that is drastically shRNAs and targeting to 16,368 human genes, were used for the screen. This different from previous arrayed screens. The advantage of our 82K pooled VSV-G pseudotyped lentivirus, prepared by the National RNAi pooled RNAi screen is that the collective surviving cells identify Core Facility in Taiwan, was separated into 10 subpools and each of these genes crucial for IAV replication but dispensable for cell pro- liferation. These genes potentially are ideal targets for antivirals. The relation between Itch and viruses has also been investigated, particularly in the field of virus release. For example, silencing Itch reduced human T-cell leukemia virus type 1 bud- ding from 293T cells (26). Moreover, Itch can facilitate Moloney murine leukemia virus release in a late domain-independent manner (27). Although we could not rule out the possible involvement of Itch in IAV release, we discovered that Itch con- tributes to the release of virus from late endosomes during the virus entry process (Fig. 3 C and D) but does not participate in the internalization of virus (Fig. S2C), nuclear import of vRNP (Fig. 3C), or RNA replication (Fig. S2B). The ubiquitin–vacuolar protein sorting system has been demonstrated to be required for influenza virus during entry into host cells (28). In the presence of the proteasome inhibitor MG132 influenza virus was seques- tered in endocytic compartment, and as a consequence its entry into the nucleus was blocked (28). Moreover, the ubiquitin– proteasome system has been shown to be important at a postfusion step of IAV infection (29). We have performed an acid bypass experiment (30) to address whether KD of Itch affects virus–cell Fig. 7. The proposed role of Itch in IAV entry. During endocytosis of IAV, Itch becomes phosphorylated and resides in late endosomes. Itch prob- fusion. The results showed that after acid PBS (pH5)-induced vi- ably mediates IAV release from late endosomes by ubiquitinating M1 to rus–cell fusion, the viral replication was lower in the Itch KD cells facilitate dissociation of viral RNA from M1 and subsequent transport of than in the control cells (Fig. S6), suggesting that Itch primarily vRNP to the nuclei.

17520 | www.pnas.org/cgi/doi/10.1073/pnas.1312374110 Su et al. Downloaded by guest on September 28, 2021 8,200-shRNA subpools was used for transduction. A total of 2.7 × 106 A549 (total volume 4.5 mL; Beckman), and overlaid with 0.5 mL 20% (wt/vol), 1 mL cells were seeded onto 150-mm plates; every two plates were used for 15% (wt/vol), 1 mL 10% (wt/vol), and 0.5 mL 5% (wt/vol) OptiPrep and then transduction with each subpool virus (MOI 0.3) to ensure the transduction of 0.5 mL HB on the top. The tubes were spun at 100,000 × g at 4 °C for 16 h 200 independent cells with the same shRNA (34). To select the shRNA-har- using a SW60Ti rotor (Beckman). After centrifugation, 0.5-mL samples were μ boring cells, the transduced cells were treated with 3 g/mL puromycin for collected from top to bottom and numbered fractions 1 to 9. Each fraction 5 d and then collected. The shRNA-harboring cells were reseeded and was subjected to Western blot analysis for the distribution of nucleolin, infected with the WSN strain of influenza virus at MOI of 1. After 2 wk, the Rab7, and actin. After Western blot analysis, fractions 2 to 7 were collected surviving cells were collected, reseeded, and reinfected with WSN as afore- mentioned to ensure that the surviving cells were resistant to IAV infection. as the endosomal fraction and fractions 8 and 9 were collected as the cy- After the second round of infection, the genomic DNA was isolated from the tosolic fraction. Viral RNA can be extracted from the collective subcellular collected surviving cells following molecular cloning procedures. Details re- fraction by using an RNA isolation kit (Roche). garding identification of shRNA and the secondary screen can be found in SI Materials and Methods. Ubiquitination Assay. HEK293T cells were transiently transfected with mam- malian expression plasmids for Myc-tagged Ub and HA-tagged M1. At 48 h Western Blot Analysis, Immunofluorescence Staining, Quantitative RT-PCR, after transfection, cells were harvested by using M-PERmammalian protein and Minireplicon Assay. Details about these procedures can be found in SI extraction reagent and centrifuged to remove the insoluble fraction. The Materials and Methods. clarified supernatant was subjected to immunoprecipitation with anti-HA affinity matrix at 4 °C for 4 h. Ub conjugates were detected by Western × 6 Subcellular Fractionation. A total of 4 10 cells were collected, resuspended blotting analysis using anti-Myc antibody. in 800 μL HB (0.25 M sucrose, 1 mM EDTA, and 10 mM Tris·HCl, pH 7.4) with protease inhibitor, homogenized using a Dounce homogenizer, and pelleted × ACKNOWLEDGMENTS. We thank the National RNAi Core Facility, Academia again at 1,000 g for 10 min. After centrifugation, the pellet (recognized as Sinica for providing lentiviral shRNAs and technical support for the RNAi the nuclear fraction) and the postnuclear supernatant (PNS) were collected screen, and Institute of Molecular Biology Imaging Core and Bioinformatics respectively. The PNS was then adjusted to a concentration of 30% (wt/vol) Core for technical assistance. We also thank Dr. Shin-Ru Shih for providing OptiPrep (Sigma-Aldrich) solution, transferred to an ultracentrifuge tube influenza A/Taiwan/3446/2002 (H3N2).

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