LIVE-CELL FLUORESCENCE MICROSCOPY OF HSV-1 CELLULAR EGRESS BY EXOCYTOSIS

The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the world’s population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.

locations on the plasma membrane, which the authors inferred was the result of viral exocytosis at preferential sites (in Vero cells, an African green monkey kidney epithelial cell line) [22][23][24][25].
A variety of egress modes have been observed with other viruses: The beta herpesvirus, human cytomegalovirus (HCMV), was recently shown to exit by bulk release -exocytosis of many particles from a larger organelle -in human foreskin fibroblast (HFF-1) cells [26]. Both flaviviruses and coronaviruses have been observed by electron microscopy to accumulate large numbers of virus particles in large intracellular organelles, but it is unclear whether these large organelles mediate bulk release or if there are subsequent intracellular sorting steps to release single virions from individual exocytosis events [27,28]. In retroviruses, HIV-1 has been observed to assemble and exit preferentially at the trailing uropod of polarized T cells [29,30], and human T-lymphotropic virus (HTLV) forms large accumulations of virions and extracellular matrix (termed "viral biofilms") on the cell surface, which may promote more efficient cell-cell spread [31][32][33][34]. Thus, the relationship between exocytosis (single particles in individual secretory vesicles, versus bulk release of many particles from a larger organelle) and accumulation at preferential locations on the cell surface following exocytosis varies according to the particular virus and cell type. But, these features of viral egress are likely important for subsequent cell-cell spread.
In the present study, we have extended our previous work on PRV to study the egress of HSV-1 particles via live-cell fluorescence microscopy. To construct a model system allowing us to visualize HSV-1 exocytosis, we engineered a recombinant strain of HSV-1 that expresses superecliptic pHluorin on an extravirion loop of the multipass transmembrane glycoprotein M (gM-pHluorin). A variant of GFP, pHluorin was developed as a means to image secretory vesicle exocytosis in a variety of cell types, including neurons [35,36]. Following secondary envelopment, pHluorin is quenched in the acidic lumen of secretory vesicles (pH of 5.2-5.7) [36,37]. When the secretory vesicle fuses with the plasma membrane to release the virus particle to the extracellular medium (pH ⇠7.5), pHluorin is dequenched and becomes brightly fluorescent, allowing the unambiguous identification of individual viral exocytosis events [16,17,37,38] ( Figure 1A).
Using this technique, we show that HSV-1 exits from infected cells by exocytosis of individual virus particles, not bulk release of many virions at once. In some cell types, viral exocytosis occurs at preferential plasma membrane sites, leading to the gradual accumulation of large clusters of virus particles, but we show that this phenomenon is cell-type dependent. Consistent with previous reports [22], mutations in viral membrane proteins gE, gI, and US9 were not essential for preferential viral egress and accumulation into clusters. To characterize the cellular mechanisms responsible for this phenomenon, we show that an exogenous kinesin microtubule motor co-accumulates at sites of cluster formation, indicating that the arrangement of the microtubule cytoskeleton likely directs virus particle transport to particular locations, resulting in preferential egress and cluster formation at these sites. Finally, using timelapse confocal imaging, we show that these large peripheral accumulations of virus particles form at sites of cell-cell contact and contribute to cell-cell spread of infection.

Results
Insertion of pHluorin into gM. To produce the recombinant strain HSV-1 IH01, we inserted the pHluorin coding sequence into the gM (UL10) gene in the HSV-1 genome by homologous recombination between a synthesized shuttle plasmid 5 and purified HSV-1 DNA. The construct was designed to insert the pHluorin moiety into the first extravirion/lumenal loop of gM ( Figure 1B). A second recombinant, HSV-1 IH02, expressing gM-pHluorin and an mRFP-VP26 capsid tag, was produced by co-infecting HSV-1 IH01 and HSV-1 OK14 [39], and purifying two-color plaques.
We confirmed the correct recombination occurred by PCR amplification and Sanger sequencing (Supplemental Material 1), and also the expression of gM-pHluorin by western blot of infected cell lysates. The western blots were probed with anti-gM and anti-GFP antibodies simultaneously (Figure 2A). Viral membrane proteins frequently produce complex banding patterns due to post-translational modifications like glycosylation and aggregation of these highly hydrophobic proteins during sample prep [40,41]. Cells infected with parental strains HSV-1 17syn + and OK14 produced major gM-immunoreactive bands near the predicted 51 kDa of native gM, whereas cells infected with the recombinant HSV-1 IH01 and IH02 strains produced bands that are immunoreactive to both gM and GFP antibodies, and shifted ⇠30 kDa, consistent with the predicted gM-pHluorin fusion ( Figure 2A).
gM-pHluorin Labels Virus Particles and Exhibits pH-Sensitive Fluorescence. To determine whether gM-pHluorin is incorporated into individual virus particles, we spotted ⇠100µl of freshly-prepared infected cell supernatants onto a glass coverslip, and imaged by fluorescence microscopy ( Figure 2B). To measure the pH sensitivity of the gM-pHluorin fluorescence, we added an excess of PBS buffer at pH ⇠6 followed by an excess of PBS buffer at pH ⇠7. gM-pHluorin incorporated into virus particles exhibited reversible pH-dependent green fluorescence, whereas the mRFP-VP26 capsid tag exhibited a non-pH-sensitive reduction in fluorescence due to photobleaching ( Figure 2B).

Virus Replication.
To determine whether the recombinant HSV-1 IH01 and IH02 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint strains replicate comparably to the parental viruses, we performed single-step growth curves ( Figure 2C) and measured plaque size ( Figure 2D) on Vero cell monolayers.
Compared to the parental HSV-1 17syn + strain, HSV-1 OK14, IH01, and IH02 exhibited a modest delay in replication at 8 hours post-infection (hpi). However, by 24 hpi, HSV-1 OK14 and IH01 had titers equivalent to 17syn + , but HSV-1 IH02 exhibited a modest <1 log defect ( Figure 2C). These data suggest that the gM-pHluorin and mRFP-VP26 fluorescent protein fusions result in a small reduction in viral replication. Consistent with these results, plaque sizes of the HSV-1 OK14 and IH01 were also reduced ( Figure 2D). Although these defects could also be explained by other mutations arising during construction, we initially characterized two different recombinants and several independent clones of each, with similar results.
Live-Cell Fluorescence Microscopy of Virus Particle Exocytosis. To investigate virus particle exocytosis with our model system, we infected Vero cells with HSV-1 IH01 at a high multiplicity of infection (MOI) to roughly synchronize viral infection, and imaged by live-cell fluorescence microscopy at approximately 5-6 hpi. This time point represents the earliest production of viral progeny, prior to the onset of cytopathic effects (CPE). To compare to our previous studies of PRV [16], we also infected PK15 cells with PRV 483, which expresses orthologous gM-pHluorin and mRFP-VP26 fusions. We identified productively infected cells by imaging in widefield fluorescence mode to detect mRFP-VP26 fluorescence in the nucleus and gM-pHluorin green fluorescence on the plasma membrane and in intracellular membranes. We then acquired timelapse movies in Total Internal Reflection Fluorescence (TIRF) microscopy mode, which excludes out-of-focus fluorescence and emphasizes particle dynamics near the adherent cell surface ( Figure 3A).
As previously reported with PRV, virus particle exocytosis events are characterized 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. by the sudden (<90 ms) appearance of green gM-pHluorin fluorescence, which then remains pucntate and mostly immobile during the time of imaging (>2-3 min) [16,17].
Exocytosis events that do not contain a particle, characterized by rapid diffusion of gM-pHluorin into the plasma membrane, are excluded from this analysis.. To quantify this process over many exocytosis events, we measured the relative fluorescence intensity at exocytosis sites for 54 sec before and after each exocytosis event, aligned all data series to a common time=0, and calculated the ensemble average over many events ( Figure   3B-C). Prior to exocytosis at time=0, the relative gM-pHluorin fluorescence remains low, consistent with pHluorin quenching in the acidic lumen of the viral secretory vesicle. At the moment of exocytosis, gM-pHluorin fluorescence increases suddenly due to dequenching at extracellular pH. Finally, the fluorescence decays gradually, which represents a combination of: 1. diffusion of gM-pHluorin that is incorporated into the vesicle membrane; 2. occasional movement of the cell or virus particle after exocytosis; 3. photobleaching. In total, we quantified 67 HSV-1 IH01 exocytosis events from over 30 individual Vero cells across six replicate experiments. These data are consistent with our previous studies of PRV exocytosis [16,17], validating that this approach works for HSV-1.

HSV-1 Particles Accumulate at Preferential Exocytosis Sites in Multiple
Cell Types. Previous studies showed that HSV-1 structural proteins and particles accumulate in large clusters at the adherent edges of Vero cells and cell-cell junctions in epithelial cells [22][23][24][25]. However, based on static fluorescence and electron microscopy images, it is unclear if virus particles gradually accumulate in these clusters due to preferential exocytosis at these sites, if large clusters are deposited at once due to bulk release (as recently observed with HCMV [26]), or if virus particles accumulate in clusters later in infection due to cell movement and rounding associated with 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint cytopathic effects. Previously, we did not observe preferential exocytosis sites or large clusters of virus particles with PRV in PK15 cells [16,17], so it was unclear whether this represents a difference in virus biology or that of host cell biology.
To better understand how these large clusters of virus particles form, we infected Vero or PK15 cells with HSV-1 IH02 and imaged at 6-7 hpi. At this time point, HSV-1 IH02 particles were beginning to accumulate in peripheral clusters in Vero cells ( Figure 4A). A representative time course is provided in supplemental material, and illustrated using a maximum difference projection ( Figure 4B, Movie S1). Maximum difference projections show where fluorescence intensity increases most rapidly, which emphasizes exocytosis events and particle movement, and deemphasizes static features that do not change during the course of imaging. In this representative Vero cell with multiple exocytosis events over time (4:01 min:sec), we observed exocytosis of particles containing green gM-pHluorin only, which we infer to be non-infectious L-particles (green boxes), and particles containing both gM-pHluorin and mRFP-VP26 capsids, which we infer to be virions (yellow circles). The diameter of detected fluorescence in these exocytosis events is consistent with diffraction-limited HSV-1 particles. Some viral exocytosis events appeared to be clustered near the cell periphery and cell protrusions, suggesting that the large peripheral clusters that appear later in infection accumulate gradually by the exocytosis of individual particles ( Figure 4B, Movie S1). In this representative exocytosis event, a virus particle (pseudocolored magenta) arrives at the location of exocytosis, is largely immobile for 10 s, and then green pHluorin dequenches, indicating exocytosis ( Figure 4B, right panel) Because we previously reported no such accumulations of virus particles with PRV in PK15 cells [16,17], we compared HSV-1 IH02 to PRV 483 in PK15 cells ( Figure   4C-D, Movie S2). In contrast to Vero cells, there appear to be no large accumulations 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. To determine whether this clustering occurs in a more biologically-relevant primary cell type, we prepared rat embryonic fibroblasts (REFs), and infected them with HSV-1 IH02 or PRV 483. In these cells, it was difficult to assess whether virus particles clustered to the same degree as in Vero cells, because REFs exhibited cytopathic effects (CPE) earlier than in the transformed cell lines; however, peripheral accumulations were observed prior to significant rounding and CPE ( Figure 5A). Similarly to Vero and PK15 cells, HSV-1 particles appeared to exit from REFs in the form of individual exocytosis events ( Figure 5B-C), again suggesting that the observed peripheral clusters accumulate gradually over time.
Because HSV-1 co-evolved in humans, we also investigated the clustering egress phenotype in human-derived cell lines: Panc-1 cells, a human pancreatic epithelioid sarcoma cell line, and a derivative of Panc-1 cells with stable RNAi knockdown of the cellular RNA helicase DHX9. The cellular functions of DHX9 remain enigmatic, but it is involved in epithelial-mesenchymal transition (EMT) in cancer biology, and has been shown to function as an antiviral factor [42]. In antiviral signaling, DHX9 expression is upregulated by IL-1 and TNF signaling, and, in turn, promotes NF-B and JAK-STAT signaling [43][44][45].

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint whereas the DHX9 knockdown cells exhibited elongated cell extensions typical of mesenchymal cell morphology ( Figure 6B) [46]. Also, consistent with the role of DHX9 as an antiviral signaling factor, DHX9 knockdown cells appeared to be more permissive to HSV-1 infection, with greater expression of gM-pHluorin and mRFP-VP26 structural proteins in our microscopy assays ( Figure 6A systems due mutations that disrupt the gE, gI, and US9 genes [50][51][52][53][54][55]. These proteins contribute to secondary envelopment, recruit microtubule motors for particle transport in multiple cell types [14,[56][57][58], and are required for axonal sorting and anterograde 11 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint axonal spread in neurons [23,50,[59][60][61][62]. However, it is not clear how mutations in gE, gI and US9 might affect egress in non-neuronal cells.
HSV-1 OK14, which is based on the 17syn + laboratory strain, expresses functional gE, gI, and US9 [39,63]. HSV-1 425 is based on the HSV-1 MacIntyre strain, which contains many polymorphisms, including mutations that disrupt gE/gI/US9 function [54,55]. Both viruses express an mRFP-VP26 capsid tag. At about 5 hpi, we manually categorized infected cells in random fields of view based on the presence of virus particle clusters at the cell periphery. Cells infected with HSV-1 425 demonstrated a roughly similar proportion of clustering compared to HSV-1 OK14 in Vero cells ( Figure 7B). These results show that gE, gI, and US9 are not strictly required for clustered egress of HSV-1. Because HSV-1 MacIntyre contains many polymorphisms compared to 17syn + , there remains a possibility that MacIntyre contains compensatory mutations that allow for a clustered egress phenotype in the absence of gE/gI/US9 function; however, these results are consistent with previous work by Mingo, et al. [22], who showed that gE is not necessary for cluster formation in Vero cells.
To compare PRV to HSV-1, and further assess the function of gE, gI, and US9 in isogenic virus strains, we also infected cells with PRV recombinants. PRV 483 is based on the common Becker laboratory strain, which expresses functional gE, gI, and US9 [64,65] . We also constructed PRV IH001, which contains the PRV Bartha vaccine strain deletion that removes the gE/gI/US9 genes, in an isogenic Becker genetic background. Both of these viruses express gM-pHluorin and mRFP-VP26. While PRV will infect and form plaques on Vero cells, we were unable to achieve sufficient levels of infection for our microscopy experiments -it is possible that the efficiency of plating of PRV in Vero cells is too low to achieve a high-MOI roughly synchronous infection in our experimental conditions. To overcome this limitation, we instead 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made and PRV exhibited clustering at the cell periphery, and gE, gI, and US9 proteins were not required for clustering. These results further reinforce the idea that this clustering effect is common to both HSV-1 and PRV, and varies by cell type. However, the polarized trafficking that is mediated by gE/gI/US9 in neurons is not essential for clustered egress in these non-neuronal cell types.
Exogenous Kinesin Microtubule Motors Co-accumulate with Virus Particles in Peripheral Clusters. To better characterize the peripheral accumulations of virus particles we observed, we identified a cellular marker that labels the "corners" and tips of cell extensions before and during virus replication and egress. A large body of literature has shown that microtubule motors mediate intracellular transport of HSV-1 particles [12,[66][67][68][69]. Based on our observations above, we hypothesized that the arrangement of the microtubule cytoskeleton might explain why virus particles accumulate at particular subcellular locations during egress. We reasoned that microtubules may be preferentially arranged with their (+) ends at these peripheral sites, leading to the preferential transport to and egress at these sites.
In preliminary attempts, we were unable to adequately resolve individual microtubules in live cell microscopy due to the high abundance of microtubules and tubulin protein throughout the cell. While microtubule (+) end binding proteins (e.g. EB1, EB3) can be used to mark the growing (+) ends of dynamic microtubules, they do not effectively mark the (+) ends of stabilized microtubules, and HSV-1 infection promotes microtubule stabilization [70][71][72]. Therefore, as a probe of microtubule arrangement, we expressed KIF1A-EmGFP, an exogenous (+) end-directed microtubule motor that accumulates at microtubule (+) ends in live cells. KIF1A is a kinesin-3 family motor that is highly expressed in neurons, where it contributes to axonal sorting and 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint transport of cellular cargoes and virus particles. However, KIF1A is not strongly expressed in most non-neuronal cells, including Vero cells [73]. We infected Vero cells with HSV-1 OK14 and an amplicon vector expressing KIF1A-EmGFP. At 6-7 hpi, we identified co-infected cells by widefield fluorescence microscopy, and found that KIF1A-EmGFP and virus particles co-accumulated at the tips of cell extensions ( Figure 8A, arrows). Figure  CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint at sites of cell-cell contact. Over the next two and a half hours, these accumulations increased from a few detectable fluorescent punctae to distinct clusters of red fluorescence along these cell-cell contacts.
In other time course examples, we observed accumulations of virus particles being transferred from one cell to another ( Figure 9A-B, Movie S3-4). Because the KIF1A-EmGFP probe labels cell protrusions prior to formation of virus particle clusters, we also imaged Vero cells co-infected with HSV-1 OK14 and the KIF1A-EmGFP amplicon vector ( Figure 8C). Consistent with the still images in Figure 9A showing that these clusters of virions can contribute to cell-cell transmission.

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Discussion
By producing an HSV-1 recombinant virus that expresses the pH-sensitive fluorescent protein, pHluorin, we have developed a live-cell microscopy assay that allows us to visualize the process of viral egress from infected cells. pHluorin is genetically fused to the viral envelope glycoprotein gM, is incorporated into virus particles, is quenched in the lumen of cellular vesicles, but dequenches upon exocytosis, allowing detection of virus particle exocytosis. We are able to detect individual virus particles undergoing exocytosis, and while this approach had been successful in previous alpha herpesvirus studies [16,17], this is the first time that this approach has been applied to the important human pathogen, HSV-1.
Altogether, these data show that the long-observed clustering of HSV-1 particles in Vero cells occurs due to preferential exocytosis of individual particles at these sites, rather than bulk release or post-exocytosis movements. In all the experimental conditions presented here, we have never observed bulk release of many virions at once. Our prior observations, that this clustering does not occur with PRV in PK15 cells, is the result of cell type differences, not virus differences. Both HSV-1 and PRV form clusters in Veros, primary REFs, and human cancer cell lines, but not in PK15 cells.
Differences in intracellular transport and secretory mechanisms may explain why the spatial distribution of viral egress varies across these cell types. We (and many others) have previously shown that alpha herpesvirus particles use cellular secretory pathways, regulated by Rab family GTPases, and recruit kinesin microtubule motors for intracellular transport to the site of exocytosis. Different cell types express different kinesin motors, different Rab GTPases, and other cell biological factors. In polarized 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint epithelial cells, distinct secretory organelles sort cargoes to the apical or basolateral plasma membrane. In neurons, vesicles containing axonal cargoes can transport into the axon, but vesicles containing somatodendritic cargoes are strongly excluded from the axon. The alpha herpesviruses have evolved to modulate axonal sorting and transport by recruiting additional kinesin motors via the viral gE, gI, and US9 proteins. However, here we show that these viral factors are not required for transport to and exocytosis at preferential egress sites in these non-neuronal, non-polarized cell types.
Instead, it appears that the morphology of the microtubule cytoskeleton may be responsible for the differences in virus particle distribution during the later stages of infection. Vero cells have been documented to form distinct focal adhesion patterns [22,74], and HSV-1 infection has been shown to alter the arrangement of microtubules [70][71][72]75]. The underlying cell biological differences in cytoskeleton arrangement between different cell types, together with the effects of viral infection (i.e. microtubule stabilization) likely account for the differences we observe in the clustered/preferential egress phenotype. Viruses. All HSV-1 or PRV strains were propagated and titered by plaque assay on Vero or PK15 cells, respectively, in DMEM supplemented with 2% FBS and 1% penicillin-streptomycin. HSV-1 17syn + and OK14 were obtained from the Lynn Enquist laboratory (Princeton University) and verified by whole genome sequencing.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint HSV-1 OK14, which expresses an mRFP-VP26 capsid tag, was previously described [39]. HSV-1 425, which is based on HSV-1 MacIntyre and expresses an mRFP-VP26 capsid tag, was a kind gift from Esteban Engel (Princeton University) [55]. PRV 483 and PRV 495, which express gM-pHluorin and an mRFP-VP26 capsid tag, were previously described [16]. PRV BaBe was obtained from the Lynn Enquist laboratory (Princeton University) [64,65]. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint replicate on its own. PRV BaBe contains a deletion in the US region encoding gE, gI, and US9 [64,65]. Following co-infection, progeny plaques were screened for green and red fluorescence. Several clones were picked, plaque purified three times, and further screened for lack of gE, gI, and US9 expression via western blot (Supplemental 2).

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint Fluorescence microscopy. All cell types were seeded at subconfluent density (⇠ 10 5 cells/dish) on glass-bottom 35mm dishes (Celltreat, Ibidi, and Mattek), incubated overnight, and then infected with HSV-1 or PRV at a relatively high MOI (>1 pfu/cell). o account for differences in the efficiency of plating between different viruses and cells, the amount of inoculum needed to synchronously infect most cells was determined empirically on a case-by-case basis using fluorescence microscopy.
MOIs ranged from 1-20 pfu/cell, as titered on Vero or PK15 cells for HSV-1 and PRV, respectively, without taking differences in efficiency of plating into account.
HSV-1 infected cells were imaged beginning at 5-6 hpi, and PRV infected cells were imaged beginning at 4-5 hpi, unless otherwise stated. Fluorescence microscopy was performed using a Nikon Eclipse Ti2-E inverted microscope in the Biodesign Imaging Core facility at Arizona State University. This microscope is equipped with TIRF and widefield illuminators, a Photometrics Prime95B sCMOS camera, a 60X high-NA TIRF objective, and objective and stage warmers for 37°C live-cell microscopy. For widefield fluorescence, a Lumencor SpectraX LED lightsource provided 470/24nm and 550/15nm excitation for green and red fluorescent proteins, respectively.
For TIRF microscopy, 488nm and 561nm lasers were used to excite green and red fluorescent proteins, respectively. Image analysis was performed using Fiji software [78]. Fluorescence microscopy images were prepared for publication using Adjust Brightness/Contrast, Reslice (to produce kymographs), and Plot Z-axis Profile (to measure fluorescence over time) functions in Fiji. Maximum difference projections were calculated as previously described [16], using the Duplicate, Stacks->Tools, Math->Subtract, and Z Project functions in Fiji.Ensemble averages of fluorescence intensity over time in a 3x3 pixel region of interest around individual exocytosis events ( Figure 3B-C, 5C, 6C) were calculated using Matlab (Mathworks).

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint For confocal microscopy, samples were imaged on a Nikon AX R laser scanning confocal microscope in the Biodesign Imaging Core facility (Arizona State University, Tempe, AZ) using a 60X 1.42 NA objective. EmGFP was excited at 488nm and mRFP at 568nm. Emissions for these channels were collected in the green and red ranges respectively. Images were acquired every 60 seconds with a 100ms exposure. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint was aspirated off and HBSS (Gibco) was added to prevent drying. Virus particles were subjected to a pH change by adding 150µL PBS at pH 6. pH was then returned to neutral by adding an excess of PBS at pH 8. Imaging was performed using widefield LED illumination and 60X magnification to detect individual virus particles.

Single
Step Growth Curve and Plaque Size Measurements. Vero cells were seeded to confluence in 35mm 6-well dishes and infected at MOI of 5 PFU/cell. The

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Figure 2. pHluorin Expression and Impact on Viral Replication
A. Western blot detecting HSV-1 gM and pHluorin. Vero cells were infected with HSV-1 17syn + , IH01, OK14, and IH02, or mock-infected. Blots were probed with primary antibodies detecting gM and pHluorin, and imaged using fluorescent secondary antibodies to detect HSV-1 gM and pHluorin simultaneously. B. gM-pHluorin is incorporated into virus particles, and exhibits reversibly pH-sensitive fluorescence. Freshly-prepared supernatants from Vero cells infected with HSV-1 IH02 were spotted onto glass bottom dishes. Particles were imaged at pH ⇠7, pH ⇠6, and pH ⇠7. A representative virus particle is shown with gM-pHluorin and mRFP-VP26. Images represent 3.6x3.6 µm. C. Single Step Growth Curve. Vero cells were infected with HSV-1 17syn + , OK14 , IH01, or IH02 and harvested at 4, 6, 8, and 24 hpi. Infections were done in triplicate for each virus at each time point. Samples were titered by serial-dilution plaque assay. Error bars represent standard deviation, asterisk represents statistical significance with p<0.05. Means of each time point were compared with one-way ANOVA in Graphpad Prism. D. Plaque size measurements. At 4 days post-infection, virus plaques were imaged, and the zone of clearance diameter was measured in Fiji software and used to calculate mean plaque sizes (n=100). Asterisk indicates statistical significance with p<0.05. HSV-17 was compared with each fluorescent strain with a Student's T-test.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint Figure 6. HSV-1 Exocytosis in Human Panc-1 Cells A-B. HSV-1 IH01 infection in human Panc-1 cells versus DHX9 knockdown cells. Compared to parental Panc-1 cells, DHX9 knockdown cells exhibited more of an elongated mesenchymal morphology, greater expression of viral structural proteins, and greater clustering at cell extensions, as assessed by fluorescence microscopy. Images are representative of 10 cells in 3 experimental replicates. Scale bars represent 10µm. C. Relative fluorescence intensity of gM-pHluorin before, during, and after exocytosis of individual virus particles of HSV-1 IH02, in DHX9 knockdown cells. Green line represents mean fluorescence and gray shading indicates standard deviation (n=13). D. Percentage of imaged cells with a clustered or distributed virus particle distribution during infection (n=100). Not significantly different (p>0.05 by Student's T test). Error bars represent standard deviation.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ;

Figure 8. A Plus End-Directed Kinesin Motor Marks Virus Clustering Sites
A. HSV-1 OK14 particles (magenta) co-accumulate with plus end-directed microtubule motor KIF1A (green) at the cell periphery. Vero cells were coinfected with HSV-1 OK14 and an HSV-1 amplicon vector expressing EmGFP-KIF1A, and imaged at 6 hpi. Accumulations (arrows) are indicated. B. In transfected cells expressing only EmGFP-KIF1A, the motor similarly accumulates at the tips of cell protrusions (arrows), in the absence of viral infection. C. Colocalization of EmGFFP-KIF1A and HSV-1 OK14 virus particles in cell protrusions that mediate cell-cell contact (arrows). Still images are from a timelapse movie of coinfected Vero cells imaged every 60 s from 5 hpi to 12 hpi. Scale bars represent 10µm.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint Figure 9. Clusters of HSV-1 Particles Form at Cell-Cell Junctions, and Contribute to Cell-Cell Transmission A. Representative still images from time lapse Movie S3 of virus particle accumulation in Vero cells. Cells were infected at high MOI with HSV-1 IH02 and imaged every 60 seconds from 4 hpi to 8 hpi. Arrows indicate areas of virus particle accumulation. B. Still images from Movie S4 time lapse of Vero cells infected with HSV-1 IH02 at high MOI. Images were acquired every 60 seconds from 11 hpi to 18 hpi. A large cluster of virus particles is transferred from an infected cell to an uninfected cell (arrows). This cell subsequently becomes infected, based on new capsid protein expression in the nucleus (N) at 17:51 hpi. A-B. Scale bars represent 10µm.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 17, 2023. ; https://doi.org/10.1101/2023.02.27.530373 doi: bioRxiv preprint