A âDriver Switchoverâ Mechanism of Influenza Virus Transport from

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A “Driver Switchover” Mechanism of Influenza Virus Transport from Microfilaments to Microtubules Li-Juan Zhang, Li Xia, Shu-Lin Liu, En-Ze Sun, Qiu-Mei Wu, Li Wen, Zhi-Ling Zhang, and Dai-Wen Pang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06926 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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A “Driver Switchover” Mechanism of Influenza Virus Transport from Microfilaments to Microtubules Li-Juan Zhang, Li Xia, Shu-Lin Liu, En-Ze Sun, Qiu-Mei Wu, Li Wen, Zhi-Ling Zhang, and Dai-Wen Pang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P.R. China

*Corresponding Author E-mail: [email protected]

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ABSTRACT: When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a “driver switchover” mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.

KEYWORDS: influenza virus, quantum dot, tracking, motor protein, microfilament, microtubule

Influenza virus is a valuable model for exploring the endocytic pathway and a paradigm for understanding virus infection. To infect host cells, the virus will experience multiple steps, including entering the cells by receptor-mediated endocytosis, moving on MFs (also called actin filaments) at the cell periphery, moving along MTs to the perinuclear region, and fusing with the endosomal membrane for genome release.1-3 But it is still unclear that how viruses switch from MFs to MTs. As the main components of cytoskeleton, MFs and MTs contribute to intracellular transport of proteins, 2


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vesicles and organelles by motor proteins traveling along them.4 Myosin superfamily members are responsible for the cargo transport on MFs, among which myoVI is the only retrograde motor protein.5 Kinesin and dynein are the anterograde and retrograde motors using MTs as tracks to transport cargos through the cytosol.6,7 Although they have been well understood individually,8-11 how the two transport systems work together for the coordinative transport of cargos remains obscure. Thus unraveling the transport mechanism of influenza virus from MFs to MTs during its infection is not only significant for fighting against influenza, but also meaningful for in-depth understanding of endocytosis. Several hypotheses about how motor proteins coordinate the transport between MFs and MTs have been proposed during the past decades. With the finding that MF- and MT-based motor proteins could be present on the same cargos, researchers hypothesized that the transportations on MFs and MTs might be simultaneously active.12-14 The physical interaction between a MF-based motor protein, myosin Va, and a MT-based motor protein, KhcU, arose another hypothesis that intracellular transport might be coordinated through direct interactions between different motor molecules.15,16 Moreover, the transport from MFs to MTs was also speculated to be a handoff process from myoVI to dynein at actin-microtubule intersections.17,18 Although these hypotheses have existed for a long time, the question is still open. It is probably due to the limitation of conventional approaches which generally solve problems through analysis of the ensemble average data of a large number of samples. However, heterogeneous samples cannot be accurately characterized by the averaged properties in complex biological environments. This conflict can be well resolved by the QD-based SVT technique, which uses microscopes to monitor the behaviors of individual QDs-labeled viruses in living cells.19-22 The outstanding brightness and photostability as well as narrow emission linewidths of QDs make the QD-based SVT a powerful tool to readily track the viruses and further study the sophisticated virus-host cell interactions.23-27 Here, QD-based SVT combined with multicolor imaging was utilized for in situ real-time 3


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monitoring of the relationships among influenza virus, myoVI and dynein on MFs and MTs. We initially investigated virus infection in cells with simultaneously labeled MFs and MTs and found that the virus moved seamlessly from MFs to MTs. To unravel the molecular mechanism behind this movement, myoVI and dynein together with MFs and MTs were labeled and their interactions during virus infection were dissected. We found that myoVI and dynein were responsible for virus transport on MFs and MTs, respectively. Interestingly, these two types of motor proteins could be attached to the same virus-carrying vesicle after virus entry and move together from MFs to MTs. Further results indicated that myoVI drove viruses on MFs with dynein on the vesicles hitchhiking, and then dynein drove the viruses along MTs with myoVI remaining on the vesicles moving together. Taken together, these results revealed a “driver switchover” mechanism of influenza virus transport from MFs to MTs, which is meaningful for the design and development of anti-influenza drugs and in-depth understanding of endocytosis.

RESULTS Virus Moving from MFs to MTs. H9N2 influenza virus and MDCK cells were used as the main model virus and cell to study how influenza virus moves from MFs to MTs. Colocalization of MFs and MTs in three dimensions in cells with enhanced green fluorescent protein (EGFP)-labeled MFs and mCherry-labeled MTs was obviously observed by three-dimensional (3D) fluorescence imaging (Figure 1A). The infection of QDs-labeled viruses in these cells was tracked (Figure 1B and C) and we found that the viruses moving on MFs switched instantly to moving along the MTs linked to the MFs without obvious pause or being hindered (n=11) (Figure 1D and Video S1). Virus movement on MFs is obviously shorter than along MTs (Figure 1E). Analyzing the dependent relationships between instantaneous velocity and time, and between mean square displacement (MSD) and time interval (∆t) indicated that during the transport process from MFs to MTs, virus movement changed immediately from slow directed motion (with D and V values of 0.0008 µm2/s and 0.051 µm/s) to fast directed motion (with D and V values 0.025 µm2/s and 0.26 µm/s) (Figure 1F and G). These 4


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results showed that although MFs and MTs are different cytoskeletal filaments, the transport of viruses between them is continuous and seamless (Figure 1H).

Figure 1. Virus moving from MFs to MTs in live MDCK cells. (A) Bright-field image of a cell, orthogonal slice views of EGFP-labeled MFs (green) and mCherry-labeled MTs (red) and the merged image. (B) Fluorescence images of QDs (705 nm)-labeled H9N2 viruses (blue), EGFP-labeled MFs (green), mCherry-labeled MTs (red) and the merged image. (C) Magnified view of the boxed region in (B). (D) Snapshots of the virus shown in (C) switching from MFs to MTs. (E) Trajectory of the virus tracked in (D). (F) Time trajectory of the instantaneous velocity of the virus. (G) Dependent relationships of MSD and ∆t of the virus. The lines are the fits to MSD = 4D∆t + (V∆t)2 + constant (D and V are the diffusion coefficient and fitting velocity, the constant term is due to noise). The cyan and pink lines in (E-G) are corresponding to virus movement on MFs and MTs, respectively. (H) Illustration of virus moving from MFs to MTs. MyoVI- and Dynein-Dependent Movements of Viruses on MFs and MTs. To investigate how influenza virus moves on MFs, QDs-labeled viruses were used to infect cells with EGFP-labeled MFs and mCherry-labeled myoVI (Figure 2A). Colocalization of viruses with myoVI traveling on MFs (n=30) was observed by multicolor imaging (Figure 2B and C; Video S2). The completely overlapped time projections of the virus, myoVI and MFs intuitively confirmed the colocalized movement of virus and myoVI on MFs (Figure 2D). Dependent relationship between instantaneous velocity and time indicated that movement of the virus was slow with speeds below 0.5 µm/s (Figure 2E), consistent with above result and the reported velocity of myoVI.28,29 Furthermore, we analyzed the dependence between MSD and ∆t. The apparent upward curve indicated that the virus movement was a directed motion with D and V values of 0.003 µm2/s and 0.14 µm/s, respectively (Figure 2F). This is consistent with the previous reports that motor protein-driven movements on cytoskeleton are directed motions.30,31 These results indicated that as the only retrograde motor protein traveling on 5


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MFs, myoVI drove influenza virus on MFs with a slow, directed motion (Figure 2G).

Figure 2. Viruses colocalized with myoVI and dynein traveling on MFs and MTs. (A) The merged fluorescence image of QDs-labeled viruses (blue), mCherry-labeled myoVI (red) and EGFP-labeled MFs (green). (B) Snapshots of a virus (pointed with white arrows) colocalized with myoVI moving on MFs. This region is indicated with a white rectangle in (A). (C) Trajectory of the virus. (D) Time projection images of the virus, myoVI and MFs, and the merged image. (E) Instantaneous velocity vs. time plot of the virus. (F) MSD vs. ∆t plot of the virus. (G) Illustration of a virus driven by myoVI on MFs. (H) The merged fluorescence image of QDs-labeled viruses (blue), mCherry-labeled dynein (red) and green fluorescent protein (GFP)-labeled MTs (green). (I) Snapshots of a virus (pointed with white arrows) colocalized with dynein moving along MTs. This region is indicated with a white rectangle in (H). (J) Trajectory of the virus. (K) Time projection images of the virus, dynein and MTs, and the merged image. Arrows in (D) and (K) indicate the tracked signals at the first time point. (L, M) Instantaneous velocity vs. time and MSD vs. ∆t plots of the virus. The dotted lines in (E) and (L) indicate the velocity of 0.5 µm/s. (N) Illustration of a virus driven by dynein along MTs. In addition, virus infection in cells with GFP-labeled MTs and mCherry-labeled dynein was imaged to study how influenza virus moves along MTs (Figure 2H). It was observed that the virus colocalized with dynein moving along MTs for a long distance toward the perinuclear region (n=220) (Figure 2I and J; Video S3), where it would release its genome.2,3 The overlapped time projections of the virus, dynein and MTs in Figure 2K showed this finding intuitively. From time trajectory of the instantaneous velocity we found that the virus was transported rapidly with speeds up to about 2 µm/s (Figure 2L). Speed fluctuation of the virus movement is related with the complex configuration of MTs.32 Dependent relationship between MSD and ∆t indicated that this movement was a directed motion with D and V values of 0.086 µm2/s and 0.23 µm/s, respectively (Figure 2M). These results confirmed that as the only MT-based retrograde motor protein, dynein drove influenza virus along 6


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MTs with a rapidly directed motion (Figure 2N). Attachment of MyoVI and Dynein Together to Virus-Carrying Vesicles. 3D multicolor imaging of viruses, myoVI, dynein and MFs showed that dynein colocalized simultaneously with viruses and myoVI on MFs in three dimensions (n=210) (Figure 3A and B). Meanwhile, simultaneous imaging of viruses, myoVI, dynein and MTs demonstrated that myoVI could also colocalize with viruses and dynein on MTs in 3D space (n=260) (Figure 3C and D). To verify this finding, cells with EGFP-labeled myoVI and mCherry-labeled dynein were infected by QDs-labeled viruses. Simultaneous colocalization of myoVI, dynein and viruses was observed from the periphery to the perinuclear region of cells (n=130) (Figure 3E; Video S4). Kymograph images of typical colocalized signals showed that myoVI and dynein could simultaneously colocalize with viruses for a long time up to about 4 min (Figure 3F). Statistic results (tMg ~ 0.46, tMr ~ 0.36, ICQ ~ 0.13) indicated that endogenous myoVI and dynein could coexist on the cargo in cells (Figure S1A and B). Besides, most viruses (about 90%) colocalized simultaneously with myoVI and dynein in infected cells (Figure S1C and D). Furthermore, viruses in different stages of transportation, such as clathrin-mediated internalization, early endosome- and late endosome- involved transports, could colocalize with both myoVI and dynein (Figure S2). Similar results were obtained for H9N2 influenza virus transport in HeLa cells and H1N1 influenza virus transport in both MDCK and HeLa cells (Figure S3-S5). These results confirmed that both myoVI and dynein were attached to virus-carrying vesicles after influenza virus entry and moved together from MFs to MTs. Since myoVI and dynein are molecular motors binding to and traveling on MFs and MTs respectively, we speculated that the two types of motor proteins on the virus-carrying vesicles just took charge of driving the viruses on their corresponding filaments (Figure 3G).

Figure 3. Simultaneous colocalization of myoVI, dynein and viruses on MFs and MTs. (A) The merged orthogonal slice view of Dylight 649-labeled myoVI (orange), Dylight 405-labeled dynein 7


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(red) and EGFP-labeled MFs (green) in a cell infected by Cy3-labeled viruses (blue) for 30 min. (B) Magnified view of the boxed region in (A) and the corresponding single orthogonal slice views of viruses, myoVI, dynein and MFs. (C) The merged orthogonal slice view of Dylight 488-labeled myoVI (orange), Dylight 405-labeled dynein (red) and Alexa 555-labeled MTs (green) in a cell infected by QDs-labeled viruses (blue) for 30 min. (D) Magnified view of the boxed region in (C) and the corresponding orthogonal slice views of viruses, myoVI, dynein and MTs. (E) Fluorescence images of QDs-labeled viruses (blue), EGFP-labeled myoVI (green), mCherry-labeled dynein (red) and the merged image. (F) Kymograph images of five typical colocalized signals of viruses, myoVI, dynein and the merge indicated with numbered colored circles in (E). (G) Illustration of the speculated transport mechanism of viruses from MFs to MTs. Two-Stage Motion of MyoVI and Dynein in the Transport Process of Viruses. Myosin-driven movement is a slow process with speeds generally below 0.5 µm/s, while the speeds of dynein-driven cargos are much higher up to several micrometers per second.33,34 In addition, cargos transported by dynein can move more than a dozen micrometers, while the displacements of myosin-driven cargos are obviously shorter.33,35,36 What’s more, the diffusion coefficients of vesicles on MFs are also smaller than those on MTs.34 Therefore, myosin- and dynein-driven movements of viruses can be distinguished by their motion behaviors together with their positional relationships with cytoskeletal filaments. To verify our hypothesis, motion behaviors of viruses and motor proteins during the infection process of viruses were analyzed, which showed that myoVI colocalized with the viruses in the periphery as well as the interior region of cells (Figure 4A). Tracking myoVI enabled us to find that its movement was a striking two-stage pattern (n=210) (Figure 4B; Video S5). In the first stage, myoVI moved slowly with speeds below 0.5 µm/s (cyan line in Figure 4C). Its accumulated distance in this period was about 2 µm (cyan line in Figure 4D). Dependent relationship between MSD and ∆t indicated that myoVI moved in a directed mode with D and V values of 0.001 µm2/s and 0.15 µm/s 8


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(cyan line in Figure 4E). Following the slow movement, the virus moved rapidly with speeds up to about 2 µm/s (pink line in Figure 4C). In this period, the virus kept moving a long distance of about 28 µm in a directed mode with D and V values of 0.01 µm2/s and 0.68 µm/s, respectively (pink lines in Figure 4D and E).

Figure 4. Two-stage movement of myoVI and dynein colocalized with viruses during virus infection. (A) Fluorescence images of QDs-labeled viruses (red), mCherry-labeled myoVI (green) and the merged image. White arrows indicate a typical colocalized signal of virus and myoVI. (B) Magnified trajectory of myoVI tracked in (A). (C-E) Instantaneous velocity vs. time, accumulated distance vs. time and MSD vs. ∆t plots of the tracked myoVI. Line colors are consistent with the trajectory colors in (B). (F) Fluorescence images of QDs-labeled viruses (red), mCherry-labeled dynein (green) and the merged image. White arrows indicate a typical colocalized signal of virus and dynein. (G) Magnified trajectory of dynein tracked in (F). (H-J) Instantaneous velocity vs. time, accumulated distance vs. time and MSD vs. ∆t plots of dynein. Afterward, virus infection in cells with mCherry-labeled dynein was studied and their obvious colocalization in different region of the cell was observed (Figure 4F). The trajectory of dynein colocalized with a virus showed that it moved from the periphery toward the interior region of the cell in a similar two-stage pattern (n=300) (Figure 4G; Video S6). Its movement in the first stage was a slow (with speeds below 0.5 µm/s), short-range (with accumulated distance of about 4 µm) and directed (with D and V values of 0.007 µm2/s and 0.069 µm/s) motion (cyan lines in Figure 4H-J). Following this slow movement, dynein moved with a fast (with speeds up to about 2 µm/s), long-range (with accumulated distance of 15 µm) and directed (with D and V values of 0.039 µm2/s and 0.11 µm/s) motion (pink lines in Figure 4H-J). These results indicated that during the transport process of viruses, both myoVI and dynein moved in a two-stage pattern. Movements in the two stages were probably driven by myoVI and dynein 9


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successively, since the slow, short-range retrograde movement is consistent with the transport characteristics of myoVI and the fast, long-range retrograde movement is dynein-dependent.34 Such a striking two-stage motion also suggested that the two types of motor proteins attached to the same virus-carrying vesicles did not exert marked effects on the function of each other to transport the virus. These results provided significant evidence for the hypothesis in Figure 3G. MyoVI-Dependent Movement of Dynein on MFs. To further characterize virus transport on MFs, infection of QDs-labeled viruses in cells with mCherry-labeled dynein and EGFP-labeled MFs was tracked and analyzed (Figure 5A). It was observed that dynein colocalized with viruses moving on MFs (n=60) (Figure 5B; Video S7). Trajectory of the tracked dynein showed that this movement was short-range (Figure 5C). Time projection images provided more intuitive evidence for the colocalized movement of dynein and viruses on MFs (Figure 5D). Instantaneous velocity vs. time and MSD vs. ∆t plots indicated that the movement of dynein was a slow, directed motion with speeds below 0.5 µm/s and with D and V values of 0.009 µm2/s and 0.10 µm/s respectively (Figure 5E and F), which was similar with the motion behaviors of myoVI moving on MFs mentioned above. Furthermore, virus infection in cells microinjected with anti-myoVI antibody was obviously inhibited by comparing with that in cells microinjected with homologous IgG (n=60) (Figure 5G and H). These results indicated that the slowly short-range directed movement of dynein in the first stage was a myoVI-dependent movement on MFs (Figure 5I), confirming the speculated transport mechanism of viruses on MFs in Figure 3G.

Figure 5. Colocalized movement of dynein and viruses on MFs. (A) Fluorescence images of EGFP-labeled MFs (green), QDs-labeled viruses (blue), mCherry-labeled dynein (red) and the merged image. (B) Snapshots of dynein (pointed with white arrows) colocalized with a virus moving on MFs. This region is indicated with a white rectangle in (A). (C) Trajectory of the tracked dynein. 10


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(D) Time projection images of MFs, the virus, dynein and the merged image. Arrows indicate the tracked signals at the first time point. (E, F) Instantaneous velocity vs. time and MSD vs. ∆t plots of dynein. (G) The merged fluorescence image of QDs-labeled viruses (red) and anti-myoVI antibody (green). (H) Statistic result of viruses in cells microinjected with IgG or anti-myoVI antibody and infected with viruses for 20 min, which was obtained from sixty cells randomly selected from four parallel experiments. The data was represented as mean ± SD. ns, *P < 0.05. (I) Illustration of the myoVI-dependent movement of dynein in the transport process of viruses on MFs. Dynein-Dependent Movement of MyoVI along MTs. Similarly, virus infection in cells with mCherry-labeled myoVI and GFP-labeled MTs was tracked and analyzed to characterize virus transport along MTs (Figure 6A). The merged image showed that myoVI could colocalize with viruses on MTs. What’s more, myoVI could move together with viruses along MTs for long-range movement with accumulated distance above 15 µm (n=120) (Figure 6B and C; Video S8). Time projection images confirmed this finding more intuitively (Figure 6D). Time trajectory of the instantaneous velocity showed that the speed of myoVI was very fast up to about 2.5 µm/s (Figure 6E). This movement turned out to be a directed motion with D and V values of 0.047 µm2/s and 0.38 µm/s, respectively (Figure 6F). These motion behaviors of myoVI were similar with the behaviors of dynein driving viruses along MTs mentioned above. What’s more, the rapid and long-range directed movement and the convergence at the perinuclear region of viruses were barely observed in ciliobrevin D (CilioD, a dynein specific inhibitor37) -treated cells (n=200) (Figure 6G, H and S6; Video S9, 10). The long-range movement of myoVI also disappeared in CilioD-treated cells (n=110) (Figure S7). These results demonstrated that the fast, long-range movement of myoVI in the second stage was driven by dynein along MTs (Figure 6I), confirming the speculated transport mechanism of viruses on MTs in Figure 3G.

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Figure 6. Colocalized movement of myoVI and viruses along MTs. (A) Fluorescence images of GFP-labeled MTs (green), QDs-labeled viruses (blue), mCherry-labeled myoVI (red) and the merged image. (B) Snapshots of myoVI (pointed with white arrows) colocalized with a virus moving along MTs. This region is indicated with a white rectangle in (A). (C) Trajectory of myoVI tracked in (B). (D) Time projection images of MTs, the virus, myoVI and the merged image. The starting positions of the tracked signals are pointed with white arrows. (E, F) Instantaneous velocity vs. time and MSD vs. ∆t plots of myoVI. (G) Fluorescence image of QDs-labeled viruses in a CilioD-treated cell with a typical trajectory indicated with a white arrow. The gray and blue lines indicate the outlines of cell plasma membrane and nucleus. (H) Instantaneous velocity vs. time plot of the virus tracked in (G). The inset is the MSD vs. ∆t plot of the virus. (I) Illustration of the dynein-dependent movement of myoVI in the transport process of viruses on MTs. DISCUSSION To successfully infect host cells, influenza virus must deliver its genome into the nucleus.1 To this end, the virus endocytosed into cells moves successively on MFs and MTs to reach the perinuclear region where the genome release occurs.2,3 As for how viruses travel between these two different cytoskeletal filaments, little research has been done to answer it. In this work, we revealed a “driver switchover” mechanism of influenza virus transport from MFs to MTs. MyoVI is the only reported actin-based retrograde motor protein which can move processively on MFs and facilitate the transport of endocytic vesicles from cell periphery.35,38,39 We observed that influenza virus could colocalize with myoVI moving on MFs by tracking individual viruses. In cells injected with myoVI antibody, the virus infection was obviously inhibited. Similar result was obtained in cells overexpressing myoVI tail,40 suggesting that myoVI could drive influenza virus on MFs. On the other hand, dynein is a MT-based molecular motor exclusively provides the driving force for fast retrograde transport, which is indispensable for many viruses to transport toward cell nucleus, such as adenovirus type 2 and 5, human immune deficiency virus type 1 and African swine 12


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fever virus.41,42 Here, we observed that dynein colocalized with influenza virus moving along MTs rapidly toward the interior region of cells. Such movement of viruses was barely observed in CilioD-treated cells, which blocked the ATPase activity of dynein specifically,37 as well as in cells injected with dynein antibody,2 confirming that dynein drove influenza virus along MTs. In our recent work, we found that clathrin-mediated endocytosis was the main route for influenza virus to infect MDCK cells.43 MyoVI is associated with clathrin-coated pits/vesicles and the ensuing uncoated endocytic vesicles.44,45 Colocalization of myoVI and dynein with viruses and clathrin suggested that not only myoVI,46 but also dynein was recruited to virus-carrying vesicles after virus entry. Further results indicated that myoVI drove the virus on MFs independently and dynein moved just by hitchhiking the vesicle. In a similar way, dynein drove the virus along MTs independently and myoVI moved together with the vesicle. This was consistent with that motor proteins drive cargos by binding to corresponding cytoskeletal filaments.5,41 In addition, we observed that influenza virus moving on MFs switched instantly to moving along the MTs linked to the MFs. And the actin and microtubule cytoskeletons are physically linked to act coordinately for intracellular trafficking,18 suggesting that the roles of myoVI and dynein exchanged at the actin-microtubule intersection to facilitate the seamless transport of viruses from MFs to MTs. Although the molecular interaction underlying the switchover is currently unclear, some other cooperation between motor proteins seems similar with that of myoVI and dynein. Dynein could move along MTs in the antegrade direction with velocity similar to kinesin.33 And influenza virus moving along MTs could rapidly turn back when it reached an intersection between MTs.32 These phenomena might be related with the coexistence and role exchange of dynein and kinesin. Besides, coexistence of myosin V and kinesin was observed on endoplasmic reticulum and speculated to be involved with vesicle switch bewteen MTs and MFs.13

CONCLUSIONS In this work, the combination of QD-based SVT and multicolor imaging enabled us to 13


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comprehensively monitor and dissect the interactions among individual influenza viruses, MFs, MTs, myoVI and dynein. And hence we revealed the “driver switchover” mechanism of influenza virus transport from MFs to MTs (Figure 7): myoVI and dynein attached together to virus-carrying vesicles drive the virus successively and independently to facilitate the seamless transport of viruses on MFs and MTs. This study has unraveled the transport mechanism of influenza virus between MFs and MTs during its infection, which is meaningful for the design and development of anti-influenza drugs. Importantly, it also indicates a switch mechanism between actin- and microtubule-dependent transports of endocytic vesicles, facilitating in-depth understanding of endocytosis.

Figure 7. Transport model of influenza virus from MFs to MTs. After virus entry, MF-based motor protein, myoVI, and MT-based motor protein, dynein, are attached together to virus-carrying vesicles. MyoVI drives the virus on MFs at the cell periphery with dynein on the virus-carrying vesicle hitchhiking. At the actin-microtubule intersection, role exchange occurs between myoVI and dynein. Dynein starts to drive the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together.

MATERIALS AND METHODS Cell Culture. Madin-Darby canine kidney (MDCK, ATCC CCL-34) and HeLa (ATCC CCL-2) cells were cultured at 37 °C in a 5% CO2 environment in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 5% fetal bovine serum (FBS, South America origin, PAN Biotech) and routinely passaged every two days. For fluorescence imaging, cells were grown on 20 mm glass-bottomed dishes (Beijing Derunfeng Technology Co., Ltd.). Virus Propagation. Influenza viruses (strain A/chicken/Hubei/01-MA01/1999(H9N2) and strain A/Puerto Rico/8/34(H1N1), China Center for Type Culture Collection) were propagated in the allantoic cavity of 10-day-old embryonated eggs (Beijing Merial Vital Laboratory Animal 14


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Technology Co., Ltd.). Purification was performed as described.47 Collected allantoic fluids containing viruses were first centrifuged twice at 7000 rmp for 60 min to remove chicken erythrocytes and then centrifuged at 110000 ×g at 4 °C for 1.5 hr in a Beckman Ty45 rotor to concentrate viruses. The viruses were suspended in phosphate buffer solution (PBS) and purified by gradient sucrose (15-60%) ultracentrifugation at 110000 ×g for 1 hr in a SW41 rotor. Viruses distributed in 37.5% sucrose solution were collected and centrifuged at 110000 ×g for 1.5 h in the Ty45 rotor to remove sucrose. Finally, the obtained viruses were suspended in PBS, aliquoted and stored at -80 °C for use. Virus Labeling. To label with QDs, viruses were firstly incubated with 10 µg Sulfo-NHS-LC-Biotin (Thermo) at room temperature for 2 hr. Unbound biotin and aggregated viruses were removed with a NAP-5 column (GE Healthcare) and a 0.22 µm pore size filter (Millipore), respectively. Biotinylated viruses at a multiplicity of infection of 5 were then incubated with MDCK cells at 4 °C for 15 min. After washing with cold PBS for 3 times, unbound viruses were removed. Then, 3 nM streptavidin-modified QDs (SA-QDs (705 nm), Wuhan Jiayuan Quantum Dots Co., Ltd.) were incubated with cells obtained above under the same condition. Redundant QDs were removed by washing with PBS. After that, the viruses were attached to cell surface and labeled by QDs.48 To dynamically track the virus infection, the cells were immediately warmed to 37 °C and imaged by a spinning-disk confocal microscope equipped with a cell culture system. Plasmid Transfection. To label dynein and myoVI, plasmids expressing mCherry-dynein, mCherry-myoVI, and EGFP-myoVI fusion proteins were constructed.44,49 Plasmids expressing EGFP-Lifeact peptide, GFP-microtubule associated protein 4 (MAP4) and mCherry-MAP4, EGFP-clathrin, EGFP-Rab5 and EGFP-Rab7 fusion proteins were used to label MFs, MTs, clathrin, Rab5 and Rab7, respectively. To transfect cells in a 20 mm petri dish, 0.5 µg of DNA and 1 µL of Lipofectamine LTX (Invitrogen) were mixed in 100 µL of Opti-MEM I reduced serum medium (Gibco). After 30 min, the DNA-LTX mixture was added to cells. Six hours later, the culture medium 15


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was changed to DMEM with 5% FBS. Thirty hours later, the cells were planted on 20 mm glass-bottomed dishes for fluorescence imaging. Immunofluorescence Labeling. Anti-myoVI (rabbit polyclonal, Santa Cruz Biotechnology), dynein heavy chain (mouse monoclonal, Abcam), CI-MPR (rabbit monoclonal, Abcam), EEA1 (mouse monoclonal, BD Biosciences) antibodies, phalloidin-FITC and α-tubulin-Alexa Fluor 555 (Beyotime Biotechnology), Dylight 405 and 649-goat anti-mouse and Dylight 488 and 649-goat anti-rabbit IgG (Abbkine) were used as follows. Cells infected with SA-Cy3 (Invitrogen)-labeled viruses for 30 min were fixed in PBS containing 4% (w/v) paraformaldehyde at room temperature for 15 min, washed with 0.1% (w/v) Triton-X 100 for 10 min and exposed in 5% (w/v) BSA at 37 °C for 30 min. After that, the cells were incubated with the mixture of anti-myoVI and anti-dynein antibodies for 1.5 hr and then the mixture of Dylight 405-goat anti-mouse and Dylight 649-goat anti-rabbit IgG for 1 hr at 37 °C. To label MFs, the cells were further incubated with phalloidin-FITC at 37 °C for 1 hr. The cells treated as described above were ready for simultaneously fluorescence imaging of viruses, myoVI, dynein and MFs. For simultaneous labeling of viruses, myoVI, dynein and MTs, the cells were infected by QDs-labeled viruses, incubated with Dylight 405-goat anti-mouse and Dylight 488-goat anti-rabbit IgG, and incubated with 0.1 mg/mL mouse IgG for 1 hr before labeling MTs with α-tubulin-Alexa Fluor 555. CI-MPR and EEA1 were labeled with corresponding antibodies using the same procedure. Microinjection and Drug Inhibition. Microinjection of anti-myoVI antibody was performed as described.50 A fluorescence microscope (Axiovert 200M, Carl Zeiss), an Eppendorf FemtoJet injection system, Eppendorf TransferMan NK2 micromanipulator (Eppendorf AG, Germany) and capillaries prepared with a model P-2000 capillary puller (Sutter Instruments Co., USA) were utilized for microinjection. Mixture of anti-myoVI antibody (0.2 mg/mL) and FITC was injected into cells planted on 20 mm glass-bottomed dishes. The cells were cultured at 37 °C for 30 min and then infected with QDs-labeled viruses. After that, the cells were ready for fluorescence imaging. 16


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CilioD (Millipore) was dissolved in DMSO to prepare a 10 mM stock solution and stored at -20 °C. Before use, a 100 µM working solution (containning 1% DMSO) was prepared with PBS. To inhibit the activity of dynein, cells were pretreated with 100 µM CilioD at 37 °C for 1 hr before the assay and the inhibitor was persistent during subsequent experiments. In the control experiment, cells were treated with 1% DMSO in PBS instead of CilioD working solution. Fluorescence Imaging. A spinning-disk confocal microscope (Andor Revolution XD) equipped with an Olympus IX 81 microscope, an EMCCD (Andor iXon DU897U single photon detector), a Nipkow disk type confocal unit (CSU 22, Yokogawa) and a cell culture system (INUBG2-PI) was used to obtain fluorescence images. Dylight 405, GFP/ FITC/ Dylight 488, mCherry/ Alexa Fluor 555/ Cy3, and Dylight 649/ 705 nm QDs/ Cy5 were excited with lasers of 405 nm, 488 nm, 561 nm and 640 nm, respectively. The fluorescence signals were separated with band-pass emission filters of 447/60 nm, 525/50 nm, 605/20 nm, and 685/40 nm, respectively. To simultaneously image multiple targets with different colors, fluorescence signals were detected with the EMCCD in the corresponding different channels separately. Image Analysis. Each frame of the video stack was denoised by processing with gauss filter. The signals of viruses and motor proteins were tracked using Imaging-Pro Plus (Media Cybernetics). The time projection image, kymograph image and orthogonal slice view were obtained by Andor iQ (Andor technology). The MSD of each trajectory was calculated for each time interval by the user-written program with MATLAB (MathWorks). The motion mode was determined by fitting MSD and ∆t to the following functions: MSD = 4D∆t + (V∆t)2 + constant (directed diffusion), MSD = 4D∆tα + constant (restricted diffusion) and MSD = 4D∆t + constant (free diffusion).30 To quantify the colocalization extent of two fluorescence signals, intensity correlation analysis (ICA) was performed with WCIF Image J.51 tMg, tMr and intensity correlation quotient (ICQ) were statistically calculated from ten randomly selected cells. The value of ICQ in the range of +0.1 ~ +0.5 indicates a strong covariance between the two images. Line profile of intracellular signals was exhibited using 17


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Imaging-Pro Plus software. Statistical Analysis. Two-Sample t-Test was utilized for statistical tests.

ACKNOWLEDGMENTS We thank professor Wei-Hua Huang for his help in the microinjection experiment. We thank Dr. Shao-Bo Wang from Wuhan Institute of Virology, Chinese Academy of Sciences, for his suggestions. This work was supported by the National Natural Science Foundation of China (No. 21535005), the National High Technology Research and Development Program of China (863 Program, No. 2013AA032204), the National Basic Research Program of China (973 Program, No. 2011CB933600), the 111 Project (111-2-10), and Collaborative Innovation Center for Chemistry and Molecular Medicine. L.-J. Zhang and L. Xia designed the study and wrote the paper; L.-J. Zhang conducted the experiments; S.-L. Liu and Z.-L. Zhang discussed the results and commented on the manuscript; E.-Z. Sun helped to construct the plasmids; Q.-M. Wu and L. Wen discussed the results; D.-W. Pang initiated the study, discussed the results and commented on the manuscript. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Additional figures and complete descriptions of figures and videos (PDF) Figure S1: Simultaneous colocalization of viruses with endogenous myoVI and dynein (PDF) Figure S2: 3D colocalization of myoVI (or dynein), viruses and clathrin (or early/late endosomes) (PDF) 18


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Figure S3: H9N2 influenza virus colocalized with myoVI and dynein during transport from MFs to MTs in HeLa cells (PDF) Figure S4: H1N1 influenza virus colocalized with myoVI and dynein during transport from MFs to MTs in MDCK cells (PDF) Figure S5: H1N1 influenza virus colocalized with myoVI and dynein during transport from MFs to MTs in HeLa cells (PDF) Figure S6: Virus infection in DMSO-treated cells (PDF) Figure S7: Movements of myoVI and virus in CilioD-treated cells (PDF) Video S1: Virus moving from MFs to MTs (AVI) Video S2: Virus colocalized with myoVI moving on MFs (AVI) Video S3: Virus colocalized with dynein moving along MTs (AVI) Video S4: Colocalization of myoVI, dynein and viruses in a live cell (AVI) Video S5: MyoVI colocalized with virus moving from the periphery to the interior region of cells (AVI) Video S6: Dynein colocalized with virus moving from the periphery to the interior region of cells (AVI) Video S7: Dynein colocalized with virus moving on MFs (AVI) Video S8: MyoVI colocalized with virus moving along MTs (AVI) Video S9: Virus infecting a DMSO-treated cell (AVI) Video S10: Virus infecting a CilioD-treated cell (AVI)

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Graphical Table of Contents 81x39mm (300 x 300 DPI)


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Figure 1. Virus moving from MFs to MTs in live MDCK cells. (A) Bright-field image of a cell, orthogonal slice views of EGFP-labeled MFs (green) and mCherry-labeled MTs (red) and the merged image. (B) Fluorescence images of QDs (705 nm)-labeled H9N2 viruses (blue), EGFP-labeled MFs (green), mCherry-labeled MTs (red) and the merged image. (C) Magnified view of the boxed region in (B). (D) Snapshots of the virus shown in (C) switching from MFs to MTs. (E) Trajectory of the virus tracked in (D). (F) Time trajectory of the instantaneous velocity of the virus. (G) Dependent relationships of MSD and ∆t of the virus. The lines are the fits to MSD = 4D∆t + (V∆t)2 + constant (D and V are the diffusion coefficient and fitting velocity, the constant term is due to noise). The cyan and pink lines in (E-G) are corresponding to virus movement on MFs and MTs, respectively. (H) Illustration of virus moving from MFs to MTs. 159x120mm (300 x 300 DPI)


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Figure 2. Viruses colocalized with myoVI and dynein traveling on MFs and MTs. (A) The merged fluorescence image of QDs-labeled viruses (blue), mCherry-labeled myoVI (red) and EGFP-labeled MFs (green). (B) Snapshots of a virus (pointed with white arrows) colocalized with myoVI moving on MFs. This region is indicated with a white rectangle in (A). (C) Trajectory of the virus. (D) Time projection images of the virus, myoVI and MFs, and the merged image. (E) Instantaneous velocity vs. time plot of the virus. (F) MSD vs. ∆t plot of the virus. (G) Illustration of a virus driven by myoVI on MFs. (H) The merged fluorescence image of QDs-labeled viruses (blue), mCherry-labeled dynein (red) and green fluorescent protein (GFP)-labeled MTs (green). (I) Snapshots of a virus (pointed with white arrows) colocalized with dynein moving along MTs. This region is indicated with a white rectangle in (H). (J) Trajectory of the virus. (K) Time projection images of the virus, dynein and MTs, and the merged image. Arrows in (D) and (K) indicate the tracked signals at the first time point. (L, M) Instantaneous velocity vs. time and MSD vs. ∆t plots of the virus. The dotted lines in (E) and (L) indicate the velocity of 0.5 µm/s. (N) Illustration of a virus driven by dynein along MTs. 128x95mm (300 x 300 DPI)


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Figure 3. Simultaneous colocalization of myoVI, dynein and viruses on MFs and MTs. (A) The merged orthogonal slice view of Dylight 649-labeled myoVI (orange), Dylight 405-labeled dynein (red) and EGFPlabeled MFs (green) in a cell infected by Cy3-labeled viruses (blue) for 30 min. (B) Magnified view of the boxed region in (A) and the corresponding single orthogonal slice views of viruses, myoVI, dynein and MFs. (C) The merged orthogonal slice view of Dylight 488-labeled myoVI (orange), Dylight 405-labeled dynein (red) and Alexa 555-labeled MTs (green) in a cell infected by QDs-labeled viruses (blue) for 30 min. (D) Magnified view of the boxed region in (C) and the corresponding orthogonal slice views of viruses, myoVI, dynein and MTs. (E) Fluorescence images of QDs-labeled viruses (blue), EGFP-labeled myoVI (green), mCherry-labeled dynein (red) and the merged image. (F) Kymograph images of five typical colocalized signals of viruses, myoVI, dynein and the merge indicated with numbered colored circles in (E). (G) Illustration of the speculated transport mechanism of viruses from MFs to MTs. 123x88mm (300 x 300 DPI)


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Figure 4. Two-stage movement of myoVI and dynein colocalized with viruses during virus infection. (A) Fluorescence images of QDs-labeled viruses (red), mCherry-labeled myoVI (green) and the merged image. White arrows indicate a typical colocalized signal of virus and myoVI. (B) Magnified trajectory of myoVI tracked in (A). (C-E) Instantaneous velocity vs. time, accumulated distance vs. time and MSD vs. ∆t plots of the tracked myoVI. Line colors are consistent with the trajectory colors in (B). (F) Fluorescence images of QDs-labeled viruses (red), mCherry-labeled dynein (green) and the merged image. White arrows indicate a typical colocalized signal of virus and dynein. (G) Magnified trajectory of dynein tracked in (F). (H-J) Instantaneous velocity vs. time, accumulated distance vs. time and MSD vs. ∆t plots of dynein. 129x133mm (300 x 300 DPI)


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Figure 5. Colocalized movement of dynein and viruses on MFs. (A) Fluorescence images of EGFP-labeled MFs (green), QDs-labeled viruses (blue), mCherry-labeled dynein (red) and the merged image. (B) Snapshots of dynein (pointed with white arrows) colocalized with a virus moving on MFs. This region is indicated with a white rectangle in (A). (C) Trajectory of the tracked dynein. (D) Time projection images of MFs, the virus, dynein and the merged image. Arrows indicate the tracked signals at the first time point. (E, F) Instantaneous velocity vs. time and MSD vs. ∆t plots of dynein. (G) The merged fluorescence image of QDs-labeled viruses (red) and anti-myoVI antibody (green). (H) Statistic result of viruses in cells microinjected with IgG or anti-myoVI antibody and infected with viruses for 20 min, which was obtained from sixty cells randomly selected from four parallel experiments. The data was represented as mean ± SD. ns, *P < 0.05. (I) Illustration of the myoVI-dependent movement of dynein in the transport process of viruses on MFs. 143x132mm (300 x 300 DPI)


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Figure 6. Colocalized movement of myoVI and viruses along MTs. (A) Fluorescence images of GFP-labeled MTs (green), QDs-labeled viruses (blue), mCherry-labeled myoVI (red) and the merged image. (B) Snapshots of myoVI (pointed with white arrows) colocalized with a virus moving along MTs. This region is indicated with a white rectangle in (A). (C) Trajectory of myoVI tracked in (B). (D) Time projection images of MTs, the virus, myoVI and the merged image. The starting positions of the tracked signals are pointed with white arrows. (E, F) Instantaneous velocity vs. time and MSD vs. ∆t plots of myoVI. (G) Fluorescence image of QDs-labeled viruses in a CilioD-treated cell with a typical trajectory indicated with a white arrow. The gray and blue lines indicate the outlines of cell plasma membrane and nucleus. (H) Instantaneous velocity vs. time plot of the virus tracked in (G). The inset is the MSD vs. ∆t plot of the virus. (I) Illustration of the dynein-dependent movement of myoVI in the transport process of viruses on MTs. 139x123mm (300 x 300 DPI)


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Figure 7. Transport model of influenza virus from MFs to MTs. After virus entry, MF-based motor protein, myoVI, and MT-based motor protein, dynein, are attached together to virus-carrying vesicles. MyoVI drives the virus on MFs at the cell periphery with dynein on the virus-carrying vesicle hitchhiking. At the actinmicrotubule intersection, role exchange occurs between myoVI and dynein. Dynein starts to drive the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. 88x58mm (300 x 300 DPI)


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