Isabelle Le Blanc

Pierre-Philippe Luyet

Véronique Pons

Charles Ferguson

Neil Emans

Anne Petiot

Nathalie Mayran

Nicolas Demaurex

Julien Fauré

Rémy Sadoul

Ronique Pons

Julien Faur

Robert G Parton

Jean Gruenberg
email: <jean.gruenberg@biochem.unige.ch

Endosome-to-cytosol transport of viral nucleocapsids

Keywords: Animals, Biological Transport, physiology, Cattle, Cell Line, Cricetinae, Cytosol, metabolism, ultrastructure, Endosomal Sorting Complexes Required for Transport, Endosomes, metabolism, ultrastructure, Epithelial Cells, virology, Fibroblasts, virology, Hela Cells, Humans, Lysophospholipids, physiology, Membrane Fusion, drug effects, physiology, Microscopy, Electron, Microscopy, Fluorescence, Monoglycerides, Nucleocapsid, metabolism, Phosphatidylinositol Phosphates, physiology, Phosphoproteins, genetics, physiology, RNA, Viral, biosynthesis, metabolism, Signal Transduction, physiology, Sorting Nexins, Time Factors, Transport Vesicles, metabolism, ultrastructure, Vesicular Transport Proteins, genetics, physiology, Vesicular stomatitis Indiana virus, physiology, Virus Replication, genetics

INTRODUCTION

Endocytosed proteins and lipids first appear in early endosomes , from where some molecules are recycled to the cell surface or 1 transported to the trans-Golgi network (TGN), while others are routed to late endosomes and lysosomes for degradation . Along the 2 -4 latter route, major progress has been made in understanding the mechanisms that control sorting of downregulated receptors. These are selectively incorporated into intra-luminal vesicles of the forming endosomal transport intermediates, which exhibit a typical multivesicular appearance. Both receptor sorting and the invagination process are regulated by PI3P signaling via Hrs, which contains the PI3P-binding motive FYVE, and by ESCRT-I, II and III complexes sequentially , . Then, multivesicular endosomal intermediates 5 6 transport their cargo of intraluminal vesicles towards late endosomes and lysosomes, where intraluminal vesicles will eventually be degraded . 6

The intra-luminal membranes of the multivesicular or multilamellar regions of late endosomes, however, also contain molecules -that are not destined for degradation. These include tetraspanins , mannose-6-phosphate receptor in transit , and MHC class II 7 8 molecules in antigen-presenting cells , as well as the poorly degradable phospholipid lysobisphosphatidic acid (LBPA). This lipid, which 9 is only found in late endosomes, plays a direct role in protein and lipid transport through this compartment . Little is known about 10 -14 the mechanisms regulating membrane invagination within late endosomes, except that LBPA itself may control the process via its putative effector Alix , a protein that also interacts with ESCRTs during HIV budding at the plasma membrane , . [START_REF] Matsuo | Role of LBPA and Alix in multivesicular liposome formation and endosome organization[END_REF] 16 17

It has long been known that vesicular stomatitis virus (VSV) and many other enveloped viruses must be endocytosed for infection to proceed, and that virions are then transported along the endocytic pathway leading to lysosomes. Beyond early endosomes , , but before the virions reach lysosomes, the acidic pH triggers the fusion of the viral envelope with endosomal membranes, releasing the nucleocapsid into the cytosol, where replication of the viral genome occurs , . We recently found that Alix down-expression with 20 21 siRNAs inhibits VSV infection , perhaps suggesting that endosomes play an active role in the infection process. Here, we followed the 15 VSV infection pathway in vivo (by monitoring viral envelope fusion, viral RNA replication and viral protein synthesis) and nucleocapsid release in vitro, to investigate whether the dynamics of endosomal membrane control viral fusion or nucleocapsid release.

RESULTS

Time-course of viral fusion

To detect fusion, VSV was labeled with self-quenching amounts of the fluorescent long-chain dialkylcarbocyanine dye Dil , . 22 23 Low, physiologically relevant amounts of VSV were then used in all our experiments (0.1 1.0 MOI, see supplementary materials). After -VSV pre-binding to the cell surface at 4 C, cells were incubated at 37 C, so that endocytosis resumed, and analyzed by time-lapse °°microscopy.

Little fluorescent signal was detected in cells kept at 4 C ( , frame t 0), even when incubated at pH 5.0 ( ), as

°Fig 1A = Fig 1B observed in vitro
, or in cells incubated at 37 C in the presence of the V-ATPase inhibitor bafilomycinA1 ( ), confirming the 22 °Fig 1B validity of the assay. By contrast, brightly fluorescent spots appeared after incubation at 37 C in the absence of the drug (

). This °Fig 1A sequence is shown both with DIC optics (DIC.avi) and Dil fluorescence (FLUO.avi) in the supplementary materials. After a short lag-time, the fluorescence emitted by individual spots increased rapidly, reaching a maximum after [START_REF] Simons | How an animal virus gets into and out of its host cell[END_REF] two characteristic fusion events that cover the time-frame observed in these experiments), much like influenza virus . Similarly, the 23 number of cells containing a fluorescent signal increased rapidly at 37 C and then leveled off (

), perhaps suggesting that viral °Fig 1D fusion was triggered before most virions reached late endocytic compartments. Indeed, it takes 30 60min at 37 C for the bulk of an . Then, however, infection was much less efficient (10 of controls), supporting the view that VSV highjacked 30 % the endocytic pathway to reach the perinuclear region more efficiently. It thus seems that, under physiological conditions, efficient infection, but not fusion, requires transport to late endosomes.

To interfere selectively with late endosome functions , , we used our monoclonal antibody against LBPA. When internalized 11 -14 31 by fluid phase endocytosis, the antibody accumulates in late endosomes upon binding to its antigen . The endocytosed antibody, but not 11 control antibodies, inhibited in a dose-dependent manner infection with VSV (

), but not Senda virus ( ) which undergoes Fig 3A high doses, further supporting the notion that most fusion events had already occurred in ECV/MVBs, prior to arrival in LBPA-containing late endosomes. When nocodazole was added to cells pre-treated with anti-LBPA antibodies, infection was further reduced when compared to the drug alone, and returned to the levels of the antibody alone upon drug wash-out (

Intra-luminal vesicles: PI3P signaling and Hrs

The unexpected findings that fusion and nucleocapsid release were sequential events, combined with the apparently crucial role of multivesicular endosomes, prompted us to investigate the possible role of endosome intraluminal vesicles in fusion and nucleocapsid delivery. During ECV/MVB biogenesis, both the formation of intra-luminal vesicles and the sorting of activated receptors within these invaginations are regulated by PI3P signaling via the PI3P effector Hrs , , . PI

A dual role for PI3P

To further explore the possible role of PI3P, we investigated whether infection was sensitive to the expression of the PI3P binding domain FYVE, using a GFP-tagged tandem FYVE construct (GFP-2xFYVE) , which we have shown to inhibit receptor sorting, but not 42 bulk transport to late endosomes . In marked contrast to PI 3-kinase inhibition, we found that 2xFYVE efficiently inhibited infection ( 43), without affecting G-protein transport to late endosomes containing LBPA (quantification in )

. We thus reasoned that PI3P, in addition to its role in the Hrs-ESCRT pathway, is perhaps also involved in nucleocapsid release from late endosomes, consistently with the presence of PI3P on late endosomes , where it may serve as a substrate for the PI3P 5-kinase 42 Fab1/PIKfyve . We thus designed an assay that monitors nucleocapsid release in vitro to study the possible role of PI3P in the process. 45

RNA export in vitro

After binding VSV to the cell surface at 4 C, the virus was endocytosed at 37 C in the absence of microtubules, and then chased into Endosomes remained latent during the assay (90 of endocytosed HRP, used as a marker of the endosomal content, remained Fig 8A

% entrapped within endosomes), indicating that RNA was not released because of some damage caused to endosomes during the in vitro incubation. Moreover, viral RNA export was inhibited by the addition of excess purified recombinant Alix or by cytosol prepared from cells overexpressing Alix ( ), consistently with our previous in vivo observations . Altogether, these observations show that our Fig 8F 15 assay measuring nucleocapsid release is valid. They also indicate that Alix controls the process directly, presumably by regulating the dynamics of late endosome internal membranes . 15

SNX16 acts as PI3P effector in nucleocapsid export

Since GFP-2xFYVE inhibited viral infection, we tested whether the tandem FYVE also interfered with nucleocapsid release in vitro. Indeed, RNA export from late endosomes was efficiently inhibited by cytosol prepared from cells overexpressing GFP-2xFYVE or, even more so, by purified, recombinant GST-2xFYVE (

). Effects were specific, since no inhibition was observed with the purified ). In addition, we confirmed that the inhibition we observed was not due to some indirect effects of the FYVE Fig 7C

domain, since RNA export was also inhibited by the addition of cytosol prepared from cells overexpressing the PI3P-binding domain PX of p40 ( ). Phox Fig 8F

To identify a possible PI3P effector that may regulates RNA export from late endosomes, we screened the distribution of proteins containing PI3P-binding domains, and found that SNX-16 was an interesting candidate. This protein is a member of the sorting nexin family and contains the PI3P-binding motive PX , . Previous studies showed that overexpressed SNX16 localizes to early and late 46 47 endosomes and that the protein plays a role in EGF receptor sorting and EGF-induced signaling , . Consistently, myc-tagged SNX16

DISCUSSION

Here, we show that the fusion of the enveloped virus VSV and the subsequent release of the viral nucleocapsid into the cytosol occur sequentially, at successive steps of the endocytic pathway. While viral fusion already occurs in ECV/MVBs, the nucleocapsid is delivered to the cytosol from late endosomes, in a process that depends on the late endosomal lipid LBPA, and its putative effector Alix/AIP1. Moreover, our data show that the delivery process is regulated by PI3P signaling via the PI3P effector SNX16. We conclude that, upon fusion of the viral envelope, the nucleocapsid is first delivered within the lumen of vesicles contained within ECV/MVBs a volume topologically equivalent to, but not continuous with, the cytosol where the nucleocapsid remains hidden. Then, the nucleocapsid travels within intraluminal vesicles to reach and penetrate late endosomes. There, the back-fusion of internal vesicles with the endosome limiting membrane delivers the nucleocapsid to the cytoplasm. Our data thus argue that such back-fusion events depend on LBPA under the control of Alix and PI3P signaling via SNX16.

Entry into the multivesicular endosome: an early one-way traffic step?

Our data show that viral fusion already occurred in ECV/MVBs, before VSV reached late endosomes. Why isn t the nucleocapsid ' concomitantly delivered to the cytoplasm? And why is transport to late endosomes then needed? All endosomes along the so-called degradation pathway, including ECV/MVBs , , , accumulate high amounts of internal membranes, and thus show a characteristic 24 26 27 multivesicular appearance, which sometimes becomes multilamellar at late stages of the pathway . Without evoking any specific 9 mechanism, one may envision that virions present in the endosomal lumen are more likely to interact with these internal membranes than with the limiting membrane, particularly at low (0.1 to 1.0) MOI, simply because the latter membranes are far more abundant.

Alternatively, VSV bound to a putative VSV receptor, perhaps glycolipids , consistently with the recent findings that VSV infection 50 51 does not depend on protein ubiquitinylation , may be selectively incorporated into internal membranes. In either situation, fusion would 41 cause the release of the nucleocapsid within an internal volume, which is topologically equivalent to but not continuous with the -cytoplasm. Nucleocapsid release into the cytoplasm and infection would then require the back-fusion of these intra-endosomal vesicles with the limiting membrane.

Evidence shows that the fate of internal vesicles and their cargo, including in particular downregulated receptors, is to be degraded in lysosomes. Both in yeast and mammalian cells, the same molecular mechanisms operate on early endosomes to sort the downregulated receptors that are destined for lysosomes into these internal membranes, and to drive the biogenesis of these internal membranes , . 5 6

And, intraluminal vesicles accumulate in the vacuole of yeast degradation mutants . Nucleocapsid delivery into the lumen of internal 5

vesicles would then be a dead-end, leading to degradation in lysosomes, rather than infection.

Late endosome membrane dynamics in animal cells

Several lines of evidence indicate that, in late endosomes of animal cells, internal membranes also contain proteins and lipids that are not destined for the lysosomes. LBPA, which is poorly degradable, accumulates within internal membranes of late endosomes in animal cells whether the lipid is present in yeast is not known. Moreover, MHC class II molecules are predominantly found within internal membranes of late endosomes (MIICs) in dendritic cells. Upon cell activation, these molecules are rapidly transported to the cell surface demonstrating that back-transport from late endosomes internal membranes can occur, at least in these cells, presumably via tubules 52

that may form at the expense of internal membranes via back-fusion . Moreover, cycling molecules of the mannose-6-phosphate receptor 9

(MPR) in transit are found within late endosome internal membranes , where they accumulate in cells containing endocytosed antibodies 8 against LBPA . Similarly, these antibodies inhibit cholesterol and nucleocapsid export (this study) from late endosomes. Finally, 11 31 anthrax toxin pore formation occurs relatively early in the endocytic pathway, but the delivery of the lethal factor to the cytoplasm requires transport to late endosomes and is inhibited by Alix downregulation . [START_REF] Abrami | Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway[END_REF] We conclude that the nucleocapsid makes use of internal vesicles to reach late endosomes, from where it is efficiently released, through the back-fusion of internal vesicles with the limiting membrane (see Model, Fig S1 supplementary materials). It thus appears that, in addition to the downregulation pathway conserved from yeast to man, animal cells have evolved a more elaborate membrane system in late endosomes for more efficient re-utilization and sorting of specialized lipid and protein. VSV may have highjacked this pathway to overcome the diffusion barrier imposed by the cortical actin cytoskeleton, and reach more efficiently the perinuclear region of the cell.

Mechanisms that control nucleocapsid delivery

Clearly, the precise mechanism that leads to nucleocapsid delivery, presumably via back-fusion of endosome internal vesicles with the limiting membranes, remains to be unraveled. However, some speculations are already possible. We find that the process depends on LBPA and its effector Alix, as well as on PI3P and SNX-16. The yeast Alix homologue Bro1p/Vps31p belongs to the same Class E sub-group of VPS genes as Vps27p/Hrs and ESCRT proteins in yeast, and Alix interacts with ESCRTs during HIV budding at the surface of T helper cells. But, most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes , where 54 the virus presumably uses the same machinery. These observations are consistent with our previous findings that LBPA itself may trigger the invagination process in late endosomes, and that Alix is a likely LBPA effector in this process . [START_REF] Matsuo | Role of LBPA and Alix in multivesicular liposome formation and endosome organization[END_REF] Interestingly, we find that Alix is required for efficient nucleocapsid release, suggesting that invagination and back-fusion are coupled mechanistically via Alix. Clearly, the precise mechanism of Alix action remains to be elucidated. As do the molecular events that regulate fission and fusion within the endosomal lumen. Indeed, it is far from clear how fission and fusion can be controlled by cytosolic machineries from the opposite side of the membrane as opposed to the role of coat proteins and SNAREs in intracellular transport. Our previously published data argue that these intraluminal fission and fusion events may depend, at least in part, on the intrinsic properties of the bilayer itself, via LBPA , . LBPA-rich membranes may have a high propensity to interact spontaneously with the limiting bilayer, 15 55 involving some sort of kiss-and-run fission and fusion events. However, proteins are likely to control the process, since, in particular, fission may remain frustrated if uncontrolled fusion occurs concomitantly, and vice-versa. Our previous data suggested that Alix negatively controls the invagination process by binding LBPA-rich membranes . Thus, a simple and na ve view is that Alix traps the 55 ï membrane intermediate in fission or fusion, by interacting with LBPA as the lipid appears on the limiting bilayer, and thereby controls the rates of both vesicle formation (invagination) and consumption (fusion). Both excess Alix and the lack of Alix are likely to deregulate this balanced process, and eventually inhibit fusion. This view is attractive, because it provides a reasonable mechanistic explanation for the coupling, which must exist between invagination and back-fusion. Indeed, if uncoupled, the internal membrane pool would disappear (uncontrolled back fusion), or the organelle collapse (uncontrolled fission).

-Further support for the existence of some molecular coupling between invagination and back-fusion comes from observations that PI3P signaling seems to regulate both the invagination process, via Hrs at earlier steps of the pathway , , , and back fusion, 5 39 40 presumably via the PI3P effector SNX16 (this study). Members of the SNX family were recently shown to contain a BAR domain , 56 which was proposed to function as a sensor of membrane curvature, based on the crystal structure of the BAR-containing protein amphyphysin , and which may also act as a GTPase binding motive . Whether SNX-16 also contains a BAR domain is not known. [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF] 56

However, one may speculate that the protein functions as a sensor or a regulator of membrane curvature at sites where invagination or back fusion occurs. It is attractive to believe that Alix and SNX16 act in tandem to integrate the invagination and back-fusion processes so that proper homeostasis of internal membranes is maintained within the endosomal lumen.

MATERIALS AND METHODS

Cells, antibodies and reagents

Baby Hamster Kidney cells (BHK) were grown and maintained as described , as was the production and purification of vesicular 24 stomatitis virus (VSV Indiana serotype)

. Recombinant Senda virus expressing RedFP was a gift from Laurent Roux (Geneva, 24 -Oligonucleotides and TaqMan probes were synthesized by Eurogentec SA (Seraing, BE).

Plasmids, RNA interference and transfection

The plasmid containing Hrs-myc was obtained from Harald Stenmark (Oslo, Norway). The production of GFP-2xFYVE and the purification of recombinant GST-2xFYVE and GST-2xFYVE were described , . The PX domains from pET32a-p40 ′ from Xeragon Inc., Huntsville, Al). BHK cells were transfected 24 h after seeding with FuGene6 using 1 g cDNA and incubated for 36h. analyzed by video microscopy, using a Leica AS MDW microscope with a CO2-and temperature-controlled stage (excitation: 549nm with a 75W Xe lamp (Leica) during 400ms). The fluorescent emission was collected by a glycerol-immersion objective with a 1.3 numerical aperture (Leica) and imaged onto a charge-coupled device (CCD) camera (Roper Scientific, CoolSnap HQM). DIC optics (Leica) was used to capture cell images before each fluorescence picture. Captured frames (exposure time, 400ms; one frame/5 min; sequence: 45 min) were processed and converted into avi movies with ImageJ software, and quantification was done manually using the ImageJ software.

Infection and replication

To monitor infection, sub-confluent BHK cells grown on glass coverslips were incubated for 1h at 4 C with VSV (0. phenol-chloroform and precipitated with 2M NaCl and 100 ethanol. Then 0.5 g total RNA was used for retrotranscription with % μ Superscript RT (Invitrogen, Scotland, UK). To retrotranscribe the genomic VSV-G RNA, we chose the oligo (

™ 5 ′
TTACCATTATTGGCCCGTCAAGCT ). Then, the transcribed DNA was subjected to TaqMan PCR using two primers and a fluorescent 3 ′ probe: forward primer: 5 -AGG CAC AGC CAT ACA AGT CAA A-3 , reverse primer: 5 -TTT GGA AGC ATG ACA CAT CCA-3

′ ′ ′ ′
probe: 5 -CCG TCT GCT TGA ATA GCC TTG TGA CTC TTG-3 . The probe was modified by addition in 5 of 6-FAM and in 3 of

′ ′ ′ ′
TAMRA. For TaqMan real-time PCR we used the ICycler.IQ (Bio-Rad laboratory, PA). When indicated, BHK cells, pre-treated or not ™ with 50 g/ml anti-LBPA antibodies, were incubated for 1h at 4 C with an HIV-1-derived vector expressing GFP and pseudotyped with μ °VSV-G protein (3 l of a 10 pfu/ml stock for 10 cells) , and then for 20h at 37 C with or without the antibody. GFP expression was Alternatively, cells were infected with 1 MOI (10 pfu/ml) recombinant Senda virus expressing RedFP for 14 hrs at 37 C. BCECF-dextran (a pH-sensitive dye) for 15 min at 37 C followed by a 30 min chase to accumulate the dye in ECV/MVBs. The endosomal °pH was measured by ratio fluorescence imaging . Briefly, the fluorescence emitted by single labeled vesicles was measured, and the 61 signal was calibrated after nigericin addition to neutralize the pH. We used the Nipkow QLC100 Real-time confocal system (Visitron systems GmbH, Puchheim, D). Coverslips were inserted into a perfusion chamber with a controlled temperature. Calibration and image processing were described . 62

Nucleocapsid release in vitro

VSV (1 g VSV/1.3 10 cells) was bound for 1h at 4 C to the surface of BHK cells, which had been pretreated for 2h with 10 M Late endosomes were loaded with VSV in vivo, and endosomal fractions were prepared. These fractions 18 g were incubated in vitro (A) [ μ ] with an ATP-regenerating (ATP), with or without cytosol for 20 min at 37 C. Then, free (cytosolic) RNA was separated from μ

  Fig 1C

  -°endocytosed tracer, including the glycoprotein G of VSV envelope , to reach late endosomes and lysosomes in the same (BHK) cells 24 25 ( illustrates the colocalization of G-protein with a fluid phase tracer in late endosomes containing LBPA after 45 min). Fig 1E Fusion occurs in endosomal transport intermediates Transport from early to late endosomes is facilitated by microtubules , , (see outline Fig S1, supplementary materials). 24 26 27 Consistently, a pulse of endocytosed tracers failed to reach late endosomes containing LBPA after a 45 min incubation at 37 C in the °presence of the microtubule depolymerizing drug nocodazole, and yet was no longer detected in early endosomes containing the Rab5 effector EEA1 (Fig S2A, supplementary materials). As expected , nocodazole also inhibited G-protein degradation, as did the 24 V-ATPase inhibitor concanamycinB (Fig S2B). Microtubule depolymerization did not affect viral fusion to any significant extent (Fig 2A-). Fluorescence ratio imaging showed that the pH of endosomes containing a pH-sensitive probe after microtubule depolymerization was B approximately 5.5 ( ), as acidic as late endosomes, and significantly more acidic than early endosomes (pH 6that fusion occurs within acidic endosomal vesicles, but before late endosomes. These vesicles correspond to the operational definition of endosomal transport intermediates, or ECV/MVBs , which accumulate endocytosed VSV G-protein or other 6 tracers in the absence of microtubules . 24 Transport to late endosomes is required for efficient infection and RNA replication Despite the lack of nocodazole effects on viral fusion ( ), infection, as monitored by G-protein synthesis, was markedly Fig 2A B reduced by nocodazole to the same extent as transport to late endosomes , and restored to control levels upon drug wash-out ( -24 27 -). This was not due to a role of the microtubules in the transport of free cytosolic capsids, since nocodazole had no effect on Fig 2D infection (Fig S3A supplementary materials) when VSV fusion was artificially induced at the plasma membrane , , in agreement with 24 29 previous observations

  ï

Fig 3B -

 3B Fig 3Bfusion at the plasma membrane. This inhibition was not due to some indirect effects of the antibody on the virions. Indeed, VSV pre-treatment with anti-LBPA antibodies did not affect VSV binding (Fig S3B, supplementary materials) or infection ( ), while an Fig 3C antibody against an exposed viral epitope efficiently blocked infection ( ) by preventing VSV binding to the cell surface (Fig 32 Fig 3C S3B supplementary materials). Strikingly, however, endocytosed anti-LBPA antibodies had little effect on viral fusion ( ), even at Fig 3D

-

  Fig 6A once fusion has occurred in wortmannin-treated cells, capsids are rapidly released into the cytoplasm.

  render infection insensitive to microtubule depolymerization ( ). The effects of the tandem FYVE were specific for Fig 7FPI3P, since overexpression of the PH domain of phospholipase C delta, which binds PI(4,5)P , had no effect on VSV infection ( 2[START_REF] Rhee | Regulation of phosphoinositide-specific phospholipase C[END_REF] Fig 7C

  °°late endosomes by allowing microtubule re-polymerization. Using this pulse-chase protocol, dextran and VSV accumulated in late endosomes, and VSV RNA minus strands then co-fractionated with late endosomes (Fig S4B supplementary materials). The viral RNA present in the fractions was not released by trypsin treatment of the membranes, indicating that capsids were present within endosomes, and not peripherally associated (Fig S4C, supplementary materials). Endosomal fractions were prepared and incubated in the assay with ATP and cytosol. Then, endosomes were separated from the cytosol (presumably containing the released viral RNA) by floatation in sucrose gradients, and RNA was quantified by RT-PCR in cytosol and endosomes. Viral RNA export from late endosomes occurred efficiently (30 of the amounts originally present in endosomes) at 37 C, but not at 4 C, and required the presence of ATP and cytosol ( % °°).

δ

  Fig 8F42

ï 2 .

 2 Switzerland), anti-Hrs antibodies from Harald Stenmark (Oslo, Norway), MDBK cells from Ernst Peterhans (Bern, Switzerland), and HIV-1-derived vector expressing GFP and pseudotyped with VSV-G from Didier Trono (Lausanne, Switzerland). For video-microscopy, BHK cells were grown in chambered coverglass (MatTeck Corporation, Ashland, MA). The monoclonal antibodies against LBPA (6C4) , BHK Lamp1 (4A1) and VSV-G (17.2.21.4 and P5D4) have been described, as well as the polyclonal antibody against VSV-Reagents were obtained from the following sources: anti-GFP monoclonal antibody from Roche Diagnostics (Rotkreuz, 24 Switzerland); anti-myc antibodies from Santa-Cruz Biotechnology, Inc. (San Diego, CA); anti-EEA1 monoclonal antibody from BD Transduction Laboratories (Lexington, KY); fluorescently labeled secondary antibodies from Jackson Immunoresearch Laboratories (West Grove, PA); rhodamine-dextran, BCECF-Dextran, DiL (1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocabocyanine perchlorate), from ′ ′ ′ Molecular Probes, Inc. (Eugene, OR); nocodazole, PMSF (phenylmethanesulfonyl fluoride), Wortmannin, Bafilomycin A1 pepstatinA, , apyrase, ATP, ProteinaseK, TPCK-treated trypsin and soybean trypsin inhibitor from Sigma Chemical Co. (St. Louis, MO); FuGene6 from Roche Diagnostics; Oligofectamine from Invitrogen, (Basel, CH), Silencer siRNA labeling Kit cy3 from Ambion, Inc. (Huntingdon, UK).

C215S 42 43 phox(PGEX4T- 1 -

 431 Marie-Claire Dagher, Grenoble, France) was amplified by PCR and introduced into pEGFPC2 after EcoRI-SalI digestion. Snx16 and pDmyc-SNX16 were obtained from Wajin Hong (Singapore); Snx16 cDNA was amplified by PCR and introduced into pEGFPC2 after KpnI-BamH1 digestion. Arginine 144, corresponding to arginine 58 in p40phox PX domain, was mutated to alanine (R144A) by site-directed mutagenesis. Hrs expression was silenced by RNAi in HeLa cells transfected with oligofectamine, according 40 to , as described , , using annealed 21-nucleotide RNA duplexes with 2-nucleotide 3 -(2-deoxy)thymidine overhangs (obtained 5815 59

Fig 1 labeling

 1 Fig 1 labeling procedure barely affected the virus infectious titer (Fig S5B, supplementary materials). Sub-confluent BHK cells in chambered

  1 1.0 MOI) in °-MEM, pH 7.4, and then for 3h at 37 C . Cells were analyzed by immunofluorescence, and infection was quantified by counting the °24 number of cells expressing newly synthesized VSV-G-protein. When indicated, microtubules were predepolymerised with 10 M μ nocodazole for 2h, and the drug remained present during infection, or cells were treated with 100nM wortmannin as in or with the 43 V-ATPase inhibitors bafilomycinA1 or concanamycinB . Alternatively, cells were pretreated overnight at 37 C with 5 or 50 g/ml 6C4 60 ° μ antibody. To quantify VSV replication, cells were infected with 0.1MOI as above. A PNS was then prepared, diluted twice in PK buffer (200mM NaCl, 200mM Tris pH 9.0, 20mM EDTA, 2 SDS), and incubated with 2 g/ l proteinaseK. Total RNA was extracted with % μ μ

  immunofluorescence or SDS gel electrophoresis and Western blotting using a monoclonal anti-GFP antibody.

  grown on glass coverslips and treated with 10 M nocodazole as above, were incubated with 10 g/ml μ μ

  were then incubated for 45 min at 37 C in GMEM containing 10 M nocodazole, washed 3X with PBS at 37 C, and ° μ °re-incubated first for 10 min in medium without drugs and then for 30 min with 100nM of the reversible V-ATPase inhibitor concanamycin B, to ensure that viral fusion, if at all, would not occur in late endosomes. ConcanamycinB was omitted during the first incubation step to limit the danger that it might interfere with transport itself , . Cells were washed 3X at 4 C with PBS, and fractions 60 63 °containing both early and late endosomes prepared . In the assay, 60 l fraction was mixed with 4.5 l concentrated salt solution 64 μ μ (0.625M Hepes pH 7.0; 75mM MgOAC , 50mM DTT), 18 l 1M KCl, 120 g HeLa cytosol and 6U apyrase or ATP regenerating 2 min at 37 C, the mixture were loaded at the bottom of a step sucrose gradient , and centrifuged at 100.000 g for 1h 27 °27 × (TLS 55 rotor). Then, the load (containing released viral RNA), and the interface (containing endosomes with non-released viral RNA) were collected. RNA was extracted from each fraction and quantified by Real Time PCR, as above. Cytosol was prepared from PNSs of untransfected or transfected HeLa cell, which had been complemented with protease inhibitors (10 M leupeptin, 1 M, aprotinin, 1 M μ μ μpepstatin), by centrifugation at 100,000 g for 1h. The supernatant (cytosol) was collected, aliquoted, flash frozen and stored at 90 C. described previously. For in vitro incubation of endosomes with 4nm BSA-gold, endosomal fractions 69 (prepared as above) were incubated with highly concentrated BSA-gold (OD 40) for 30 min at 4 C as described for nucleocapsid 520 = °release.

Figure 1 ) μ 7 (

 17 Figure 1 VSV fusion BHK cells were incubated with low amounts (0.3 MOI) Dil-labeled VSV at 4 C. The temperature was raised to 37 C, and cells were (A) °°imaged by time-lapse confocal microscopy. The figure shows frames captured at the indicated time, and arrows point at fluorescent spots that represent fusion events. Cells were treated as in (A) in the presence (baf) or absence (ctrl) of 1 M bafilomycinA1. The number of cells (B) μ containing fused viruses was counted after 35 min at 37 C (post-infection: p-i ). Alternatively, cells with bound virions were incubated at pH °5.0 at 4 C, and fusion events were immediately counted and expressed as a percentage of the control at 37 C. The figure shows two °°(C) representative examples of the endocytic time-course of viral fusion (as in A): the emitted fluorescence was quantified after tracking each spot in the sequence. ( The number of cells treated as in (A) containing fused viruses was counted at the indicated times during the 37 C D) °incubation, and is expressed as a percentage of the total cell number. ( Excess virus (50 g/1.3x10 cells) were pre-bound to the cell surface E) μ

Figure 2

 2 Figure 2 Microtubule-dependent transport Viral fusion was studied as in in cells pre-treated with 10 M nocodazole for 2h (the drug was present throughout the experiment), (A) Fig 1A μ and frames were captured at the indicated time. After treatment with or without nocodazole as in (A), the number of cells containing fused (B) viruses was counted at the indicated times during the 37 C incubation. Values are expressed as a percentage of the untreated control after °35min, as in . After microtubule depolymerization as in (A), BCECF-dextran was endocytosed for 10 min at 37 C and chased for Fig 1B (C) °35min. The pH of individual endosomes was measured by BCECF-dextran fluorescence ratio imaging. The histogram shows the pH distribution of 550 endosomes with the means SD indicated on the figure. VSV (1MOI) was bound at 4 C to the surface of BHK cells ±

Figure 3 Figure 4

 34 Figure 3 VSV infection requires transport to late endosome Cells were preincubated with 5 or 50 g/ml anti-LBPA ( LBPA) antibody or 50 g/ml mouse IgG (mIgG) for 14h. Cells were then (A) μ α μ infected with 1MOI VSV for 3h, fixed, analyzed by fluorescence microscopy (example shown with 50 g/ml antibody). Infected cells were μ quantified as in . Cells treated as in A with 50 g/ml anti-LBPA antibodies were infected with recombinant Senda virus Fig 2D (B) μ ï expressing RedFP (1MOI) for 14h, and analyzed by fluorescence microscopy. Infected cells are expressed as a percentage of the untreated controls typically, 25 of the cells were infected in controls. VSV was preincubated for 1h at 4 C with antibodies against the indicated -%

  α

Figure 5 Figure 6 6 -

 566 Fig 3A After 20h (20h PI), GFP expression was analyzed by SDS gel electrophoresis and Western blotting using anti-GFP antibodies, or anti-annexin2 antibodies as a loading marker (upper panel). Alternatively cells were analyzed by immunofluorescence using a monoclonal anti-GFP antibody, followed by rhodamine-labeled anti-mouse antibodies. Treated cells show a punctate staining pattern, corresponding to the endocytosed anti-LBPA (revealed by the secondary anti-mouse antibody), but not the diffuse GFP staining pattern visible in the controls. Number of experiments: A B, 4; C, 3. Bar, D: 4 m μ

Figure 7 Figure 8

 78 Figure 7 Hrs and PI3P The expression of Hrs was silenced using fluorescently labeled siRNAs (Hrs fluo-siRNAs), to identify transfected cells (B). The inset (A B) in (A) shows the Western blot of cells treated with Hrs siRNAs using anti-Hrs antibodies. Cells were then infected with VSV with or without nocodazole (noc), as in , and analyzed by fluorescence microscopy to identify cells containing labeled siRNAs and cells expressing Fig 2D VSV-G. The star shows an infected cell that did not contain siRNAs, for comparison. Infected and non-infected cells were quantified as in Fig . Cells were transfected with GFP-2xFYVE or GFP-PH for 36h, infected with VSV (1MOI), and analyzed by fluorescence microscopy 2D (C) to identify transfected cells. Infected and non-infected cells were quantified as in . After transfection with GFP-2xFYVE, as in (C), Fig 2D (D) excess VSV (50 g/1.3 10 cells) was bound to the cell surface. The virus was labeled with anti-VSV antibodies, followed by secondary μ × 7 antibodies and then endocytosed for 45 min, as in . Cells were fixed, labeled with anti-LBPA antibodies and analyzed by Fig 1E double-channel fluorescence. The number of cells where both markers colocalized was counted and is expressed as a percentage of the control. Viral fusion was quantified as in in cells overexpressing GFP-2xFYVE, as in C. RNA replication was quantified as (E) Fig 1A B-

  °endosome-associated RNA by floatation in a sucrose gradient, and VSV RNA minus strand was quantified by TaqMan-RT-PCR. RNA export is expressed as the ratio of free RNA released in the presence of ATP and cytosol over the negative controls without cytosol and ATP. (B) VSV endocytosed for 45 min (as in ) in cells expressing Snx16-myc was analyzed by fluorescence microscopy; VSV-G colocalization Fig 1E with LBPA was quantified as in . After expression of WT Snx16-myc or Snx16 -myc, HeLa cells were processed for Fig 7D (C) R144A immunofluorescence using antibodies against myc and Lamp1 (upper panels, double immuno-fluorescence) or myc alone (lower panel). (D) The assay was as (A) with cytosol prepared from cells overexpressing Snx16 (Snx16 cyt) or Hrs (Hrs cyt), or with control cytosol supplemented with 0.5 g purified recombinant Snx16 (Snx16). Cells expressing or not Snx16, Snx16 or Hrs were infected with VSV μ , and analyzed by fluorescence microscopy; infected and non-infected cells were quantified as in . The assay was as in (A) Fig 2D (F) with cytosol prepared from cells overexpressing GFP-2xFYVE (2xFYVE cyt), Alix (Alix cyt), the GFP-PH domain of PLC (PH cyt) or the ™ GFP-PX domain of p40 (PX cyt). Alternatively, the assay was carried out with control cytosol supplemented with 0.5 g purified, phox μ recombinant Alix, GST-2xFYVE (2xFYVE) or GST-2xFYVE (2xFYVE ). VSV RNA export was expressed as a percentage of the C125S C125S positive control, to facilitate comparison between different experiments. Number of experiments: A, 6; B E 3; F, 4. Bar, C: 2,5 m.

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

[START_REF] Von Schwedler | The Protein Network of HIV Budding[END_REF]

Acknowledgements:

We would like to thank Brigitte Viaccoz, Marie-Claire Velluz and Marie-H l ne Beuchat for expert technical assistance, Uli Laemmli and é è Christoph Bauer (Bioimaging platform, Frontiers in Genetics NCCR, Geneva) for help with light microscopy and imaging. We are very grateful to Kiyoshi Miwa and Didier Trono for providing us with concanamycin B, and HIV-1-derived vector pseudotyped with VSV-G, respectively. We also wish to thank Gisou van der Goot and Marc Fivaz for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (J.G.), the International Human Frontier Science Program (J.G and RGP), and the Australian National Health and Medical Research Council (RGP). ILB was supported by the award of the Bettencourt Foundation price (Prix des Jeunes Chercheurs), by the French Cancer Research Association (ARC) and by the Roche Foundation. VP was supported by the Roche Foundation and EMBO.