Lipid Tales of Viral Replication and Transmission

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Review

Lipid Tales [149_TD$IF]of Viral Replication and Transmission Nihal Altan-Bonnet1,* Positive-strand RNA viruses are the largest group of RNA viruses on Earth[8_TD$IF] and [152_TD$IF]cellular membranes are [153_TD$IF]critical [154_TD$IF]for all aspects of their life cycle, from entry and replication to[1_TD$IF] exit. In particular, membranes[12_TD$IF] serve as platforms for replication and as carriers to transmit[15_TD$IF] these viruses to other cells,[156_TD$IF] the latter either as [157_TD$IF]an envelope [158_TD$IF]surrounding a single virus or as the vesicle [159_TD$IF]containing a population of[13_TD$IF] viruses[14_TD$IF]. [160_TD$IF]Notably, many animal and[1_TD$IF] human viruses [16_TD$IF]appear [162_TD$IF]to [163_TD$IF]induce [164_TD$IF]and exploit phosphatidylinositol 4-phosphate[165_TD$IF]/cholesterol-enriched [16_TD$IF]membranes [167_TD$IF]for [168_TD$IF]replication, whereas many plant and insect-vectored animal viruses [169_TD$IF]utilize phosphatidylethanolamine[170_TD$IF]/cholesterol-enriched [17_TD$IF]membranes [167_TD$IF]for [172_TD$IF]the [173_TD$IF]same [174_TD$IF]purpose; [175_TD$IF]and [176_TD$IF]phosphatidylserine-enriched [17_TD$IF]membrane carriers are widely used [178_TD$IF]by [179_TD$IF]both [180_TD$IF]single and [18_TD$IF]populations of viruses[182_TD$IF] for transmission. Here[18_TD$IF] I discuss the [183_TD$IF]implications [154_TD$IF]for viral pathogenesis and therapeutic development[184_TD$IF] of this remarkable convergence on specific membrane lipid blueprints for replication and transmission. Introduction Single positive-strand or so-called ‘sense-strand RNA’ [(+) ssRNA] viruses encompass many important pathogenic and nonpathogenic human, animal, and plant viruses, including [186_TD$IF]poliovirus (PV), [187_TD$IF]hepatitis C virus (HCV), [140_TD$IF]rhinovirus, [18_TD$IF]norovirus, Dengue virus, Zika virus, [189_TD$IF]hoof and [190_TD$IF]mouth [19_TD$IF]disease virus, [192_TD$IF]tobacco [193_TD$IF]mosaic virus, and [194_TD$IF]tomato [195_TD$IF]bushy [196_TD$IF]stunt virus (TBSV). Perhaps more than any other family of viruses, they rely on intracellular membranes for all aspects of their life cycle, from entry and replication to exit [1,2]. Once the ssRNA slips into the cytoplasm of the host cell, it is rapidly translated into structural and nonstructural proteins. The nonstructural proteins, which comprise the machinery for viral RNA replication, are assembled on intracellular organelle membranes [132_TD$IF]which [197_TD$IF]henceforth become platforms for viral RNA synthesis. Even though preexisting cellular organelles are harnessed, viral proteins, either alone or in conjunction with coopted host proteins, rapidly transform the protein and[1_TD$IF] lipid composition and structure of these organelles to ones that are optimized for replication. Furthermore, after replication and assembly into new viral particles,[20_TD$IF] (+) ssRNA viruses can co-opt membranes as carriers to be transported out of cells nonlytically and be transmitted to other susceptible hosts. These newly synthesized viruses can emerge as single membrane enveloped particles or as populations of viral particles, transported together to the next cell within membrane-bound vesicles [3,4]. In particular, the latter, which enables trafficking of viral populations en masse from cell to cell, can significantly enhance viral infectivity and replicative fitness [3,4].

[21_TD$IF]Replication [198_TD$IF]on Membranes Viral replication organelles originate from nearly every eukaryotic intracellular membrane. PV, Coxsackievirus B3 (CVB3), [140_TD$IF]rhinovirus, [18_TD$IF]norovirus, West Nile virus, Dengue virus, Zika virus, HCV, and [187_TD$IF]hepatitis A virus (HAV) typically harness secretory pathway membranes, including the endoplasmic reticulum (ER), the Golgi apparatus, and the trans-Golgi network (TGN)[2_TD$IF], whereas [19_TD$IF]rubella virus, Brome [193_TD$IF]mosaic virus (BMV), and TBSV use the plasma membrane, the outer

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Trends RNA viruses utilize membranes with specific lipid blueprints for replication and transmission. PI4P, PE, and cholesterol are critical lipids exploited by many RNA viruses to facilitate replication. Membranes are critical for both enveloped and nonenveloped RNA viruses for their exit and transmission. Membrane vesicles can transport multiple viral particles together from one cell to another, a novel type of transmission that enhances infection efficiency. PS, a potent anti-inflammatory molecule, is frequently enriched in the membranes exploited for exit and transmission.

1 Laboratory of Host-Pathogen Dynamics, National[7_TD$IF] Heart, Lung and Blood[15_TD$IF] Institute, National Institutes of Health, Bethesda, MD, USA

*Correspondence: [email protected] (N. Altan-Bonnet).

http://dx.doi.org/10.1016/j.tcb.2016.09.011 Published by Elsevier Ltd.

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membrane of the mitochondria, and peroxisomal membranes, respectively [[20_TD$IF]1,2]. Viral replication machinery is assembled on the cytoplasmic[201_TD$IF] membrane leaflet of [20_TD$IF]these [203_TD$IF]organelles [204_TD$IF]prior to the commencement of viral RNA synthesis. Replication on the surface of [205_TD$IF]membranes has several distinct advantages over replication in the cytoplasm. First,[24_TD$IF] being on a membrane[206_TD$IF] restricts viral protein movement[25_TD$IF] to [207_TD$IF]two [208_TD$IF]dimensions, thus potentially saving time [179_TD$IF]when proteins[209_TD$IF] need to find each other [136_TD$IF]to form a complex[210_TD$IF]. This can provide a significant advantage [21_TD$IF]in getting replication going during the early stages of infection[18_TD$IF] when viral proteins are [142_TD$IF]not [21_TD$IF]abundant in the host cell [[213_TD$IF]5]. Second, [214_TD$IF]binding [149_TD$IF]to membrane lipids can [215_TD$IF]be a way either to directly or indirectly regulate viral protein enzymatic activity[216_TD$IF], the latter by influencing protein positioning on the membrane [[217_TD$IF]6–[218_TD$IF]8]. Lastly, the architecture of the membranes[219_TD$IF] themselves can facilitate replication by generating pockets in which to concentrate[27_TD$IF] and protect [20_TD$IF]viral machinery from host innate immune defenses [[21_TD$IF]2,9–[2_TD$IF]11]. Electron microscopic studies of replication organelles[23_TD$IF] have revealed complex vesiculotubular structures with both positive and negative curvature membranes that bear little resemblance to the parental organelles from which they [24_TD$IF]were derived [[25_TD$IF]9,12,13]. Host ESCRT proteins have critical roles in shaping replication organelle membranes during BMV and TBSV infections. For example, the combined actions of host ESCRT and BMV viral 1a proteins generate the negative curvature needed to form spherules in the outer mitochondrial membrane, where subsequently BMV replication machinery is concentrated and BMV RNA is synthesized [[26_TD$IF]14]. Similarly, in TBSV infections, the peroxisomal membrane is remodeled into spherules by a combination of[27_TD$IF] activities from viral p33 and host [28_TD$IF]ESCRTI and ESCRTIII protein complexes [[29_TD$IF]15]. Furthermore, host reticulon proteins are frequently recruited by viral machinery to stabilize the resulting positive curvature domains generated by negative curvature induction [[230_TD$IF]16–18]. Notably, the size of an individual membrane spherule is partly regulated by the size of the genomic RNA replicating within it. For example, spherules with a diameter of 66 nm are generated when replicating fulllength 4.8-kb genomic TBSV RNA, whereas smaller spherules are generated with smaller replicating RNA molecules [[29_TD$IF]15]. Hijacked host proteins also appear to be virally exploited to regulate access to[231_TD$IF] the viral replication complexes, and protect [23_TD$IF]them [23_TD$IF]from [234_TD$IF]host [235_TD$IF]innate immune defenses. In HCV and HAV infections, nuclear pore complex proteins have been shown to span replication organelle membrane tubule necks and impede the nuclear localization signal (NLS)-lacking innate immune effectors RIG-I and MDA5 from accessing replication sites [[236_TD$IF]19], while letting through NLS-containing viral replication proteins and accessory host nuclear factors, including NF90 and NF110 [[237_TD$IF]19,20]. This type of gating may also help regulate the levels of replication complexes at the organelles.

Phosphatidylinositol 4-Phosphate: Discovery of a Panviral Lipid [238_TD$IF]Regulating Replication Replication organelles differ from their parental host organelles in terms of not only structure, but also protein and lipid composition [239_TD$IF]and many different (+) ssRNA viruses appear to exploit a common replication organelle lipid blueprint. [240_TD$IF]This reliance among different viruses on a common membrane blueprint for replication was first observed using live cell imaging methodologies in combination with lipidomic and proteomic approaches[241_TD$IF] in PV, [24_TD$IF]CVB3, and HCV infections [21]. Investigations into the lipid composition of PV, CVB3, and HCV replication organelles[30_TD$IF] revealed membranes that [243_TD$IF]were highly enriched in PI4P lipids[24_TD$IF] both in vitro [[245_TD$IF]21] and in vivo [22] and moreover that PI4P lipids were required for viral RNA synthesis by all three viruses [21,22]. Significantly, these findings [246_TD$IF]were [247_TD$IF]later also confirmed for[31_TD$IF] replication [248_TD$IF]organelle membranes formed during [140_TD$IF]rhinovirus, [249_TD$IF]echovirus, Aichi virus, [250_TD$IF]enterovirus 71, and [251_TD$IF]encephalomyocarditis virus (EMCV) infections, among others [[25_TD$IF]23–[253_TD$IF]26] (Figure 1). Studies of PV and CVB3 infections revealed PI4P to be generated in situ at replication organelles, through viral recruitment and stimulation of pre-existing pools of host Type III

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PE dependent replicaon

PI4P dependent replicaon

Tomato bushy stunt virus Cucumber necrosis tombus virus Cymbidium ring spot virus Carnaon ring spot virus Nodamura virus Flock house virus

Poliovirus Coxsackievirus Rhinovirus Aichi virus Enterovirus 71 Hepas C virus Encephalomyocardis virus

Mitochondria

Peroxisome ER/Golgi/TGN Viral RNA

Viral RNA polymerase

Nucleus PE lipids Sterol

PI4KIIIα or PI4KIIIβ

PI4P lipids Sterol

Figure 1. Positive Single-Stranded RNA [(+) ssRNA] Viruses Exploit Phosphatidylethanolamine (PE) and Phosphatidylinositol 4-Phosphate (PI4P) Lipid-Enriched Organelles for Replication. Many plant- and insect-vectored animal (+) ssRNA viruses utilize the surface of PE and[1_TD$IF] cholesterol[127_TD$IF] enriched membranes for genome replication. Organelles with pre-existing pools of PE and cholesterol, such as mitochondria and peroxisomes, are[128_TD$IF] virally hijacked and further enriched in these lipids. By contrast, many human and animal (+) ssRNA viruses appear to rely on PI4P and cholesterol-enriched membranes for replication. Upon infection, these viruses hijack host Type III PI4 kinases[129_TD$IF] and vesicular and[130_TD$IF] non-vesicular cholesterol-trafficking pathways to transform the secretory pathway membranes [endoplasmic reticulum (ER), Golgi, and trans-Golgi network (TGN)] into[13_TD$IF] so-called replication organelles [132_TD$IF]which [13_TD$IF]subsequently become highly enriched in PI4P and cholesterol. [134_TD$IF]Both [135_TD$IF]the [136_TD$IF]PE/[2_TD$IF]cholesterol and[137_TD$IF] the PI4P[3_TD$IF] cholesterol-enriched membranes facilitate viral RNA synthesis by helping dock and concentrate viral replication proteins; by stimulating viral enzymatic reactions; and by generating high curvature membrane pockets that can concentrate and segregate viral replication machinery[138_TD$IF] away from the host innate immune defenses.

phosphatidylinositol-4-kinase b (PI4KIIIb) enzymes [[254_TD$IF]21,27][25_TD$IF], [256_TD$IF]resulting in a nearly [257_TD$IF]six-fold increase in whole-cell PI4P levels within a matter of hours post infection. PI4KIIIb[258_TD$IF] enzymatic activity was also the source of PI4P lipids in [259_TD$IF]the [260_TD$IF]replication organelles formed during rhinovirus [[261_TD$IF]7,24,25] and Aichi virus infections [[26_TD$IF]23], whereas[32_TD$IF] another member of the PI4K family, PI4KIII/, [263_TD$IF]largely [264_TD$IF]was responsible for generating the PI4P lipids at[3_TD$IF] replication organelles[265_TD$IF] formed in HCV or EMCV infected cells [[26_TD$IF]22,26,28–[267_TD$IF]30]. It is [268_TD$IF]noteworthy that all of these viruses have evolved to harness type III PI4 kinases rather than type II PI4 kinases[34_TD$IF]. Although both type II and type III enzymes catalyze PI4P production from phosphatidylinositol, type II enzymes are smaller and differ from type III enzymes in their catalytic domains [[269_TD$IF]27]. [270_TD$IF]One possibility for this preference is that the larger type III kinases afford more interaction sites for regulation by viral proteins[271_TD$IF], [27_TD$IF]and [273_TD$IF]access [274_TD$IF]to [275_TD$IF]a [276_TD$IF]wide range of host effectors [27_TD$IF]including [278_TD$IF]the multi-functional scaffolding 14-3-3 protein and the Rab11 [279_TD$IF]GTPase [280_TD$IF][27], that enable viruses to recruit other [281_TD$IF]proteins [28_TD$IF]and lipids [283_TD$IF](e.g., cholesterol[284_TD$IF], see below), to replication organelle membranes. In addition, while there is some overlap in subcellular distribution, type II enzymes

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mainly localize to, and regulate, host endocytic [285_TD$IF]pathway membranes [[269_TD$IF]27] [286_TD$IF]which may provide a smaller[23_TD$IF] surface area for establishing viral replication platforms compared [287_TD$IF]to secretory pathway membranes, not to mention [28_TD$IF]properly functioning endocytic pathways[39_TD$IF] may be vital for the viral life cycle [289_TD$IF]by [290_TD$IF]maintaining access to extracellular nutrients and other[291_TD$IF] necessary factors.

[29_TD$IF]Viral [293_TD$IF]Interior Design: Generating PI4P[40_TD$IF]-Enriched Replication [294_TD$IF]Organelles In all eukaryotic cells, secretory pathway membranes are highly dynamic, as demonstrated by the Golgi apparatus: at steady state, [295_TD$IF]this organelle is maintained through continual membrane flux, anterograde and retrograde, between itself and the ER. Arf1, a small RAS family GTPase, has a major role in the maintenance of[41_TD$IF] secretory pathway[296_TD$IF] organelles. Upon activation (by swapping GDP for GTP), it recruits to[42_TD$IF] the ER, Golgi, and TGN[297_TD$IF] membranes a variety of effectors, including[298_TD$IF] the lipid-modifying enzymes PI4KIIIb and phospholipase D,[29_TD$IF] the membrane-shaping coat proteins and their adaptors[43_TD$IF] coatomer, clathrin and GGA proteins, and[30_TD$IF] numerous cytoskeletal proteins and regulators[4_TD$IF] [301_TD$IF][31–33]. [302_TD$IF]Their combined activities[45_TD$IF] create specialized membrane domains, [30_TD$IF]which [304_TD$IF]sort and [305_TD$IF]traffic [306_TD$IF]secretory [307_TD$IF]cargo and [308_TD$IF]resident [309_TD$IF]organelle proteins,[310_TD$IF] to help generate and maintain the secretory pathway [[31_TD$IF]31–[312_TD$IF]33]. While the [31_TD$IF]spatio-temporal regulation of effector selection and recruitment is still not well understood[46_TD$IF], PV and CVB3 3A proteins were found to modulate this process [[314_TD$IF]21,34]. Early in infection, when viral 3A levels are low, PV and CVB3 use pre-existing Golgi and TGN membranes as sites of viral RNA synthesis[315_TD$IF] likely because their[47_TD$IF] PI4P lipids levels are[128_TD$IF] already relatively high compared with other cellular organelles [[316_TD$IF]21,35]. However, increasing 3A production (due to positive feedback between viral RNA synthesis and viral RNA translation) results in selective enhancement[317_TD$IF] in the recruitment of PI4KIIIb enzymes to Arf1containing membrane sites, which include the[318_TD$IF] ER exit site proximal ER–Golgi intermediate compartment, the Golgi apparatus, and the TGN, all the while suppressing the recruitment of Arf1-dependent membrane coat proteins and many other [319_TD$IF]Arf1-effectors [[25_TD$IF]21]. Consequently, the dynamics of membrane trafficking among the secretory organelles [320_TD$IF]becomes profoundly disrupted and the Golgi and TGN are eventually disassembled, with the Golgi membranes being resorbed back into the ER. Without coat proteins and other Arf1 effectors to sort and traffic[48_TD$IF] secretory cargo and rebuild[49_TD$IF] secretory organelles, the membranes emerging from ER exit sites instead become organelles 321_TD$IF]s[ pecialized [32_TD$IF]for replicating viral RNA[32_TD$IF]: highly enriched in host PI4KIIIb enzymes[324_TD$IF], PI4P lipids [325_TD$IF]and viral replication proteins[18_TD$IF] including[49_TD$IF] 3A[51_TD$IF] and the viral[326_TD$IF] RNA-dependent RNA polymerase [[25_TD$IF]21]. Remarkably ectopic expression of viral 3A [327_TD$IF]protein alone is sufficient to bring about this dramatic reorganization of the secretory pathway membranes[328_TD$IF] in uninfected cells [[25_TD$IF]21] and [329_TD$IF]appears to be mediated [30_TD$IF]through physical interactions among 3A, Arf1,[31_TD$IF] GBF1 [32_TD$IF](the guanosine exchange factor[52_TD$IF] of Arf1[3_TD$IF]) [[314_TD$IF]21,34], the Golgi adaptor protein acyl-coenzyme A-binding domain containing protein 3 (ACBD3) [[34_TD$IF]36–[35_TD$IF]38], and PI4KIIIb [[36_TD$IF]21,39]. [37_TD$IF]Notably in HCV infections, the secretory pathway is largely unaffected and[38_TD$IF] it is the viral NS5A proteins[209_TD$IF] that recruit host PI4KIII/ enzymes directly to[53_TD$IF] domains[39_TD$IF] of the ER that lie outside of ER exit sites[54_TD$IF] [340_TD$IF][22,30]. An unexplored area of investigation is what, if any,[49_TD$IF] impact[5_TD$IF] disrupting secretory pathway dynamics and/or high levels of PI4P lipids [341_TD$IF]have on cellular physiology. In chronic or persistent viral infections, such as those observed with CVB3 or HCV, replication at some level is maintained over many years with higher [342_TD$IF]than [34_TD$IF]normal PI4P [34_TD$IF]levels reported in [345_TD$IF]vivo in [346_TD$IF]infected tissues [[347_TD$IF]22]. While[58_TD$IF] secretory activity is largely unchanged for HCV, the degree of in vivo secretory perturbation is unknown for PV, CVB3, and rhinovirus infections[348_TD$IF], though it has been speculated that[18_TD$IF] if present, it may be beneficial to[349_TD$IF] these viruses by inhibiting the cell surface presentation of MHC class I molecules to the immune system [[350_TD$IF]40]. Alternatively, [351_TD$IF]these viruses may be able to maintain secretory activity in vivo and still obtain sufficient quantities of PI4P lipids to sustain replication by[60_TD$IF] setting up[26_TD$IF] negative feedback [352_TD$IF]loops among PI4K enzyme recruitment and/or activation, secretory suppression, and viral RNA synthesis and/or viral protein translation pathways, where the latter[61_TD$IF] become attenuated when PI4P levels get too high and/or secretion is blocked.

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Type III PI4 Kinases as Panviral Therapeutic Targets The exploitation of host Type III PI4 kinases by multiple different viruses makes them attractive panviral therapeutic candidates [[35_TD$IF]27] and targeting[26_TD$IF] host[51_TD$IF] rather than[26_TD$IF] viral [354_TD$IF]proteins decreases the likelihood of resistance developing quickly. Indeed, in in vitro studies, acutely inhibiting PI4KIIIb or PI4KIII/ kinases with small molecules such as PIK[35_TD$IF]93 or AL-9, depleting them from the host cell, overexpressing kinase dead mutants, or ectopically expressing phosphatases to lower PI4P levels, have all been shown to successfully block viral RNA synthesis [356_TD$IF]across many different types of [357_TD$IF]infections [[358_TD$IF]21–30,41,42], while not significantly affecting cell viability. The latter [359_TD$IF]may [360_TD$IF]be due to compensation of host PI4P needs by redundant enzymatic activities [361_TD$IF]of other PI4 kinase family members[63_TD$IF] [[269_TD$IF]27]. In contrast to in vitro studies, results from in vivo studies have been harder to interpret. In some studies, animals treated with PIK93 or closely related small molecules, while protected from viral replication, have manifested gut epithelial toxicity and lymphoid cell proliferation, while other studies have found little to no adverse effects [[362_TD$IF]41–43]. Some of the toxicities observed appear to be due to crossreactivity of PIK93 or its structural analogs with Class III PI3 kinases and Class 1B PI3Kg, whose catalytic sites are closely related to those of type III PI4 kinase [[36_TD$IF]44]. Given the potential [364_TD$IF]benefits of developing a single panviral therapeutic that can be effective against many types of viral [357_TD$IF]infections, it will be important to continue to develop and test new type III PI4K inhibitors, such as the recently developed class of PI4KIIIb inhibitors that have significantly less crossreactivity with other lipid kinases [[36_TD$IF]44].

Other Panviral Lipids Regulating Viral Replication Phosphatidylethanolamine Many plant- as well as some insect-vectored animal (+) ssRNA viruses appear to be dependent on PE-enriched membranes for replication, and depletion of PE[365_TD$IF], much like what was reported for PI4P, significantly inhibits viral RNA synthesis [[36_TD$IF]8,45,46][367_TD$IF] (Figure 1). The plant viruses TBSV, Cymbidium Ring Spot virus, and Cucumber Necrosis Tombus virus all generate[3_TD$IF] PE-enriched replication organelles from primarily peroxisome membranes, while Carnation Ring Spot virus and the insect-vectored animal viruses Nodamura and Flock House exploit the mitochondrial outer membrane for this purpose [[368_TD$IF]8,46]. In uninfected cells, both peroxisomes and mitochondria are already high in PE, but [369_TD$IF]their PE levels are further enhanced upon infection, largely as a consequence of PE redistribution from other subcellular organelles. Although the mechanism of this redistribution is unclear,[370_TD$IF] in TBSV infections a single[64_TD$IF] protein, p33, is sufficient to accomplish it [[371_TD$IF]45], akin to the [372_TD$IF]role [37_TD$IF]played by 3A proteins in PV and CVB3 infections [[25_TD$IF]21] and NS5A proteins in HCV infections [[374_TD$IF]22,30]. PE is[375_TD$IF] also one of the most abundant lipid species in bacterial membranes[376_TD$IF] [47]. To date, only a few bacterial (+) ssRNA viruses have been discovered, mostly members of the Leviviridae family. While the Leviviridae Qb replicase can replicate RNA in solution in vitro[37_TD$IF] [48], it will be important to determine in vivo whether its activity is [378_TD$IF]dependent [379_TD$IF]on membranes (i.e., bacterial cytoplasmic membrane) and particularly PE lipids. Although the pool of investigated plant- and insectvectored animal (+) ssRNA viruses is small, findings point to a preference for PE-enriched membranes as replication platforms (and, hence, use of mitochondrial and peroxisomal membranes) (Figure 1). It remains to be determined whether PE preference is more widespread among these viruses and whether [380_TD$IF]there [381_TD$IF]is an evolutionary proximity between these viruses and current or ancestral bacterial (+) ssRNA viruses. Cholesterol Frequently, cholesterol is also found enriched along with PI4P or PE at replication organelles [[382_TD$IF]7,49–[38_TD$IF]51]. Cholesterol is well known to regulate protein and lipid packing, and recent investigations have revealed that it is required for the formation of large (i.e., more than a few nanometers) and stable phosphoinositide- or PE-enriched lipid domains [[384_TD$IF]52]. Unlike phospholipids, cholesterol does not have a large head group protruding from the membrane[324_TD$IF]. [385_TD$IF]It is

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hypothesized to act as a spacer to reduce the intermolecular electrostatic repulsive forces among phosphoinositide lipids [[386_TD$IF]52,53] as well as [387_TD$IF]order the acyl chains of PE lipids, thereby stabilizing the negative curvature membrane domains induced by [38_TD$IF]them [[389_TD$IF]54] (Figure 1). Consistent with this idea, depleting cholesterol from PV, CVB3, or rhinovirus replication organelles[390_TD$IF] has resulted in smaller PI4P-enriched replication organelles and[137_TD$IF] has significantly reduced viral RNA synthesis [[391_TD$IF]7]. In PV-, CVB3-, [140_TD$IF]rhinovirus-, and [249_TD$IF]echovirus-infected cells, >90% of the plasma membrane cholesterol pools are redistributed to the replication organelles within 2 h of infection [[391_TD$IF]7]. These cholesterol pools are internalized and transferred to Rab11-positive recycling endosomes, which then traffic to replication organelles to deliver [392_TD$IF]their cholesterol cargo [[39_TD$IF]7,51]. Rab11 is itself an effector of PI4KIIIb in[20_TD$IF] eukaryotic cells [[394_TD$IF]55] and physical complex formation between Rab11 and PI4KIIIb [359_TD$IF]was found to be highly enhanced (>[395_TD$IF]four-fold) over uninfected cells during infection with PV or CVB3, even though the levels of either Rab11 or PI4KIIIb [243_TD$IF]were unchanged [[391_TD$IF]7]. Thus, by [396_TD$IF]enhancing PI4KIIIb [397_TD$IF]recruitment to replication organelles and exploiting the [398_TD$IF]preexisting affinity between PI4KIIIb and Rab[39_TD$IF]11 [55], these viruses [40_TD$IF]can target cholesterol-filled recycling endosomes to ER-based replication organelles [[401_TD$IF]7]. These findings raise the possibility of [402_TD$IF]developing [403_TD$IF]novel [40_TD$IF]types [405_TD$IF]of therapeutics that[406_TD$IF] rather than targeting catalytic domains, instead target and disrupt the interaction interface between PI4KIIIb and Rab[407_TD$IF]11. [408_TD$IF]This [409_TD$IF]type [410_TD$IF]of [41_TD$IF]strategy [412_TD$IF]may [413_TD$IF]also [41_TD$IF]result [415_TD$IF]in less[67_TD$IF] in vivo[416_TD$IF] toxicity. Nonvesicular cholesterol transfer proteins, Osh, and oxysterol-binding protein (OSBP), also have roles in enriching for cholesterol at viral replication organelles [[417_TD$IF]56]. Osh proteins have been shown to transfer cholesterol to TBSV replication sites and stimulate replication [[418_TD$IF]50,57] and OSBP depletion leads to some inhibition of HCV, PV, CVB3, and [140_TD$IF]rhinovirus replication [[419_TD$IF]24,58]. OSBP proteins have been previously demonstrated[420_TD$IF], in uninfected cells[421_TD$IF], to swap PI4P for cholesterol[42_TD$IF] in order to transport cholesterol against its concentration gradient [[423_TD$IF]59]. [42_TD$IF]In PI4Pdependent viral infections, OSBP proteins may also [425_TD$IF]play [426_TD$IF]a [427_TD$IF]similar [428_TD$IF]role, [429_TD$IF]fine-tuning cholesterol[430_TD$IF] transfer between Rab11 endosomes [431_TD$IF]and replication organelles[70_TD$IF]. Further support for feedback between PI4P and cholesterol levels during infection comes from inhibition of PI4KIII/ recruitment in HCV-infected cells, which leads to a decrease in both PI4P and cholesterol pools at the replication organelles [[432_TD$IF]49].

Lipid Regulation of Viral RNA Synthesis [71_TD$IF]Although positive-strand RNA replication occurs on membranes,[43_TD$IF] surprisingly many viral replication components, including the RNA-dependent RNA polymerases (RdRp) of PV, CVB3, [140_TD$IF]rhinovirus, Flock House virus, and TBSV, are soluble proteins. Thus,[72_TD$IF] specific lipids may facilitate membrane recruitment of these proteins [[25_TD$IF]21]. [43_TD$IF]Indeed investigations with PV and CVB3 RNA polymerases[435_TD$IF] have revealed a highly selective binding site for PI4P lipids [[25_TD$IF]21] and studies with TBSV replication proteins have also shown that binding of the viral p33 and p92 [436_TD$IF]replicase [437_TD$IF]components to PE helps them form a membrane-associated complex [[438_TD$IF]8,46]. In addition, the clustered PI4P or PE lipid-enriched domains may facilitate concentration and even polymerization of viral replication proteins that, in turn, can enhance viral RNA synthesis. [439_TD$IF]Supporting this idea, electron microscopic studies of isolated replication organelles from PV-infected cells[40_TD$IF] have revealed 2D membrane-associated viral RNA polymerase arrays [[41_TD$IF]60]. In addition, PI4P and PE have been shown to modulate viral enzymatic activities. The autocatalytic cleavage of[49_TD$IF] RNA-priming PV 3CDpol[150_TD$IF] [327_TD$IF]protein, [42_TD$IF]into the polymerase (3Dpol) and protease (3C), is significantly attenuated when its bound to PI4P lipids [[391_TD$IF]7]. Given that RNA priming is [4_TD$IF]required for RNA elongation, docking 3CDpol[43_TD$IF] proteins on PI4P lipids may be a mechanism by which[75_TD$IF] the rate of [45_TD$IF]self cleavage (in cis or trans) [410_TD$IF]is [46_TD$IF]regulated to ensure that there will always be sufficient levels of primed viral RNA for elongation. Similarly,[47_TD$IF] while binding to PE[56_TD$IF] has been shown to directly

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stimulate the enzymatic activity of[48_TD$IF] the TBSV [436_TD$IF]replicase [49_TD$IF]complex and facilitate [450_TD$IF]its association with viral RNA [[451_TD$IF]8,45,46],[76_TD$IF] binding to phosphatidylglycerol [452_TD$IF]inhibits [453_TD$IF]it [[45_TD$IF]8]. While ESCRT and reticulon proteins have important roles in sculpting replication organelles,[45_TD$IF] specific lipids [456_TD$IF]such as PI4P or PE, either alone or through interactions with host and viral proteins, can also [457_TD$IF]shape [458_TD$IF]membranes and induce organelle morphologies that are[60_TD$IF] conducive to replication. For example, in PI4P lipid-enriched membrane monolayers, the large head group: acyl chain ratio of PI4P lipids [459_TD$IF]tend to bend the monolayer away from the head groups [431_TD$IF]and form positive curvature membrane domains, whereas[78_TD$IF] PE lipids with their small head groups bend the membrane monolayer towards each other to form negative membrane curvature domains (Figure 1) [[460_TD$IF]61]. Along with host PI4P-binding FAPP2 proteins, which are found at replication organelles, the PI4P-enriched positive curvature tubular domains may be further pronounced and stabilized [[461_TD$IF]62] to generate complex 3D membrane-bound spaces that[462_TD$IF] can both concentrate [325_TD$IF]and spatially segregate viral replication machinery. Indeed, depleting either PI4P lipids or FAPP2 proteins from PV[463_TD$IF], CVB3 or HCV-infected cells dramatically alters the morphology of replication organelles and inhibits viral replication [[46_TD$IF]21,22,63].

Widespread Role for Membranes in Facilitating Nonlytic Viral Exit and Transmission Convention has long dictated that enveloped viruses (i.e., those with a membrane around their nucleocapsids) escape the cell by budding out from the surface or by budding into an exocytic compartment and that cell lysis is not a prerequisite for[3_TD$IF] exit and/or transmission to the next cell [465_TD$IF](although [46_TD$IF]it may [467_TD$IF]still [468_TD$IF]occur due to stress[79_TD$IF] or being cleared by the immune system[469_TD$IF]). [470_TD$IF]By contrast, until the discovery of an enveloped form of HAV [64], for so-called ‘nonenveloped viruses’ [471_TD$IF]which [472_TD$IF]included HAV, PV, CVB3, [473_TD$IF]rhinovirus, and [47_TD$IF]norovirus (among many others), cell lysis [475_TD$IF]had always been considered [476_TD$IF]as a prerequisite for exit and transmission[82_TD$IF]. HAV after replication and assembly on [47_TD$IF]ER-derived membranes [478_TD$IF]was [479_TD$IF]reported to be internalized in multivesicular bodies (MVB) and released to the extracellular environment in small vesicles known as ‘exosomes’ (Figure 2) [[480_TD$IF]64]. [481_TD$IF]The MVB–plasma membrane pathway[84_TD$IF] exists in uninfected cells [[482_TD$IF]65] and is an important [483_TD$IF]mechanism for [48_TD$IF]intercellular communication[86_TD$IF] [[485_TD$IF]65,66]. [486_TD$IF]By contrast to the conventional MVB[487_TD$IF] to lysosome maturation pathway, this[8_TD$IF] secretory MVB pathway is[48_TD$IF] however poorly understood and much of what is known is focused on the [489_TD$IF]machinery regulating [490_TD$IF]exosome [491_TD$IF]formation at the [492_TD$IF]MVB (e. g., ESCRT and tetraspanin proteins) rather than [493_TD$IF]MVB [49_TD$IF]plasma [495_TD$IF]membrane [496_TD$IF]targeting [497_TD$IF]and [498_TD$IF]fusion [[482_TD$IF]65]. Soon after the discovery of an exosomal HAV, other nonenveloped viruses, including [187_TD$IF]hepatitis E virus (HEV), PV, CVB3, and [140_TD$IF]rhinovirus [[49_TD$IF]3,67–[50_TD$IF]69], were also[501_TD$IF] all found to be released from cells in extracellular vesicles and all before cell lysis [[502_TD$IF]3,68]. [302_TD$IF]These [503_TD$IF]recent discoveries of nonenveloped viral particles[504_TD$IF] being released from cells nonlytically and transmitted in membranebound vesicles, similar to bona fide enveloped viral particles, [50_TD$IF]highlights [142_TD$IF]the universally central role[506_TD$IF] played by membranes in viral exit and transmission. While HEV, similar to HAV, appears to harness secretory MVBs [[507_TD$IF]64,67], PV, [508_TD$IF]CVB3, and [140_TD$IF]rhinovirus all exploit the host secretory autophagy pathway to exit cells (Figure 2) [[502_TD$IF]3,68–[509_TD$IF]70]. Autophagy is primarily a degradative pathway, triggered in times of cellular stress, where cytoplasmic cargo is selectively captured within double-membrane autophagosomes, which can originate from a variety of cellular sources, including the ER [[510_TD$IF]71,72], mitochondria [[51_TD$IF]73], and even the plasma membrane [[512_TD$IF]74]. Autophagy is a highly regulated process with cargo receptors for specific types of cargo [[513_TD$IF]72]. In canonical autophagy, once autophagosomes have formed, they typically fuse with lysosomes and their contents are degraded into protein, lipid, and carbohydrate building blocks for the cell [[510_TD$IF]71,72]. However, in PV- and CVB3-infected cells, the newly assembled viral particles at the PI4P[92_TD$IF]/[2_TD$IF]cholesterol-rich replication organelles are captured within ER-derived autophagosomes [3] and, rather than fusing with lysosomes, they are instead trafficked to, and fuse with, the plasma membrane [3]. PV-carrying autophagosomes appear to

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Viral cooperavity and enhanced replicaon

Hep A virus Hep E virus Infecon with mulple viral genomes

Poliovirus Coxsackievirus Rhinovirus

ER

PS lipids

Dengue virus West Nile virus Chikungunya virus

HIV Ebola virus Marburg virus VSV virus

PS lipids block anviral inflammaon

Figure 2. RNA Viruses Exit Cells in Phosphatidylserine (PS)-Lipid Enriched Membranes as Single or Populations of Viral Particles. Both enveloped and nonenveloped viruses exploit PS-enriched membranes to be released from cells either as single particles [Dengue, West Nile, Ebola, vesicular stomatitis virus (VSV), HIV] or as populations [Poliovirus (PV), Coxsackievirus B3 (CVB3), Rhinovirus, Hepatitis A virus (HAV), and Hepatitis E virus (HEV)]. PS lipids are obtained by budding into the endoplasmic reticulum (ER); by budding off from the plasma membrane; or by being captured in secretory autophagosomes and multivesicular bodies (MVB) and subsequently released to the outside in extracellular vesicles. The PS-enriched membranes can[139_TD$IF] potentially suppress a widespread, adaptive immune response against the virus. In addition, enhanced infection efficiency is observed by viruses traveling as populations within vesicles (e.g., PV, CVB3, and [140_TD$IF]rhinovirus [3]) [14_TD$IF]as [142_TD$IF]the vesicle [143_TD$IF]enables [14_TD$IF]delivery [145_TD$IF]of multiple viral genomes simultaneously into the host cell,[146_TD$IF] thus perhaps allowing subsequent cooperative interactions[6_TD$IF] among viral quasispecies[147_TD$IF], that benefit replication[148_TD$IF], to take place.

lack at least one protein from the SNARE machinery, syntaxin 17, which is required for[93_TD$IF] fusion[514_TD$IF] with lysosomes [3]. Fusion with the plasma membrane results in [51_TD$IF]the [516_TD$IF]release [517_TD$IF]of vesicles (the inner membrane-bound [518_TD$IF]compartments of the former [519_TD$IF]autophagosomes) filled with infectious virions to the extracellular space. The secretory autophagy pathway is also exploited in uninfected cells to secrete IL1b [[520_TD$IF]75,76], synuclein [[521_TD$IF]77], amyloid b protein [[52_TD$IF]78], bone morphogens and mineralizing agents [[523_TD$IF]79], and even[94_TD$IF] intact organelles[524_TD$IF] such as mitochondria, the latter [52_TD$IF]from reticulocytes undergoing maturation into red blood cells [[526_TD$IF]80]. Nevertheless, almost nothing is known regarding [527_TD$IF]which [528_TD$IF]cellular machinery regulates whether an autophagosome becomes degradative or secretory and how[529_TD$IF] eventual plasma membrane targeting and fusion is accomplished [[530_TD$IF]75]. [531_TD$IF]Furthermore whether both secretory and degradative pathways [532_TD$IF]can [53_TD$IF]coexist [534_TD$IF]within [97_TD$IF]infected cells and [53_TD$IF]which molecular cues [536_TD$IF]are present on viral proteins [537_TD$IF]for selective recognition by one pathway over [538_TD$IF]the other remains to be determined. Once the vesicles carrying viral cargo are released, the subsequent infection is virus receptor dependent and not simply mediated by membrane–membrane fusion between the vesicle and host cell because, at least for HAV, PV, CVB3, and [140_TD$IF]rhinovirus, pre-incubating[49_TD$IF] susceptible cells with virus receptor-blocking antibodies [539_TD$IF]inhibits infection [[540_TD$IF]3,64]. This suggests that the vesicle membranes are ruptured, during or after uptake into[541_TD$IF] an endocytic compartment within the new host cell, [542_TD$IF]as [543_TD$IF]prior [54_TD$IF]to [54_TD$IF]that viruses are[84_TD$IF] resistant to neutralizing antibodies [[546_TD$IF]64,81], the

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latter a potentially significant immune evasion [547_TD$IF]benefit for this mode of transmission [548_TD$IF]for nonenveloped viruses. Nevertheless, this raises the[10_TD$IF] question of how this membrane is[10_TD$IF] ruptured. Recent evidence [549_TD$IF]suggest endosomal lipases and cholesterol transfer proteins, such as the Niemann-Pick transporter,[50_TD$IF] as having a role in the process [[51_TD$IF]81]. In addition, the viral cargo itself could facilitate [52_TD$IF]the rupture [53_TD$IF]of its own vesicle as many nonenveloped viral capsid proteins, [54_TD$IF]such as the [5_TD$IF]adenovirus protein VI, [56_TD$IF]rotavirus VP5, and the [57_TD$IF]rhinovirus and HAV[58_TD$IF] VP4 proteins, under acidified conditions (such as those that would be encountered within endosomes), undergo conformational changes that are sufficient to penetrate and disrupt membranes [[59_TD$IF]82–[560_TD$IF]85].

Phosphatidylserine Lipids Have a Panviral Role in Facilitating Viral Infectivity [104_TD$IF]PS lipids are a major component of the[561_TD$IF] eukaryotic host[562_TD$IF] cell lipid repertoire. After de novo synthesis from PC and/or PE at the ER, they can remain in the ER,[105_TD$IF] be transferred to[49_TD$IF] mitochondria [[563_TD$IF]86,87] or to the inner leaflet of the plasma membrane, the latter in exchange for PI4P lipids [[564_TD$IF]88–[56_TD$IF]90]. From the plasma membrane, they can then be trafficked to[56_TD$IF] a variety of other organelles including endosomes, MVBs, and lysosomes. PS lipids are present in the [567_TD$IF]membranes of many bona fide enveloped (+) and (–) ssRNA viruses, including Dengue, West Nile and Chikungunya, Ebola, Marburg and [568_TD$IF]vesicular [569_TD$IF]stomatitis viruses (VSV); [570_TD$IF]retroviruses, including HIV; and even DNA viruses, such as Vaccinia[106_TD$IF] (Figure 2) [[571_TD$IF]91]. [572_TD$IF]Remarkably [573_TD$IF]PS lipids were also[574_TD$IF] found enriched in[49_TD$IF] membranes carrying nonenveloped viral particles, including[57_TD$IF] both the secretory autophagosomes carrying PV, CVB3, and [140_TD$IF]rhinovirus populations, as well as the extracellular vesicles derived from them [3] and[576_TD$IF] likely the exosomes carrying HAV and HEV (Figure 2) [[57_TD$IF]86]. While[107_TD$IF] the PS lipids of secretory autophagosomes [578_TD$IF][3] may originate: from increased PS synthesis at the ER and/or replication organelles; from increased trafficking of PS from the plasma membrane to the ER and/or replication organelles, possibly with the Rab11 carriers containing sterols [[391_TD$IF]7]; and/or by decreased transfer of PS from the ER to the plasma membrane as a consequence of high PI4P levels in the former[579_TD$IF] (due to viral recruitment of PI4K enzymes [[25_TD$IF]21][580_TD$IF]). The presence of PS lipids in the membranes [37_TD$IF]shuttling [581_TD$IF]both nonenveloped and enveloped viruses suggests[108_TD$IF] a significant role for these lipids in viral pathogenesis. Indeed, PS lipids are well-known potent anti-inflammatory agents that modulate the immune responses of the host [[582_TD$IF]92]. As part of normal cell turnover in the body, apoptotic cells are recognized and phagocytosed by both professional phagocytes (e.g., macrophages and monocytes) and nonprofessional phagocytes (e.g., epithelial cells and fibroblasts). Apoptotic cells are sensed by phagocytes because of their exposure of PS lipids from the inner leaflet to the outer leaflet of the plasma membrane. These exposed PS lipids are recognized by PS receptors on phagocytes, either through direct binding, such as with the TIM1 receptor, or by indirect binding via bridging molecules to AXL receptors [[582_TD$IF]92–[583_TD$IF]94]. This results in the phagocytic uptake of PS-enriched membranes and, in professional phagocytic cells, such as macrophages, triggers an anti-inflammatory response [[584_TD$IF]93,94]. Growing evidence suggests that PS-enriched[58_TD$IF] membranes surrounding enveloped and nonenveloped viruses exploit this pathway (so-called ‘apoptotic mimicry’ [[586_TD$IF]95][109_TD$IF] to be internalized [[587_TD$IF]3,91]. Indeed, blocking TIM, AXL, and other PS receptors or masking PS with PS-binding proteins, such as Annexin V, significantly inhibits binding, endocytosis, and infection by Chikungunya, Dengue, West Nile Virus, Ebola, Marburg, [58_TD$IF]arenaviruses, PV, CVB3, and [140_TD$IF]rhinovirus [[587_TD$IF]3,91]. [589_TD$IF]But in addition to facilitating internalization, the panviral exploitation of PS lipids may be to suppress inflammation and the wider immune [590_TD$IF]responses to the virus. In vivo studies in the context of the immune system will be needed to test this hypothesis. [591_TD$IF]Finding PS lipids[10_TD$IF] in[1_TD$IF] membranes [158_TD$IF]surrounding so many different viruses also provides an opportunity for the development of panviral therapeutics targeting these lipids. Bavituximab, a PS-targeting monoclonal antibody, has shown success in blocking infectivity of PS-enriched

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enveloped virions, such as Ebola and Lassa fever viruses,[592_TD$IF] both in in vitro assays and in small animal studies [[593_TD$IF]96,97]. Further studies, including those with nonenveloped virions, will be needed to evaluate its full clinical potential.

En masse Viral Transmission and Increased Infection Efficiency due to Extracellular Membrane Vesicles [594_TD$IF]Exploitation of membranes for exit by nonenveloped viruses has also led to[49_TD$IF] re-examination of the long[59_TD$IF] and widely-held belief that viruses only travel between and infect cells as single infectious units. [596_TD$IF]Challenging this idea, each secretory autophagosome-derived[597_TD$IF] extracellular vesicle was[598_TD$IF] remarkably found to carry a population of infectious PV, CVB3, or [140_TD$IF]rhinovirus particles [59_TD$IF]rather than a single virus particle [[60_TD$IF]3,4,66]. Given that a single PV[12_TD$IF] is  30 nm in size, an autophagosome of 500-nm diameter can, in theory, contain many hundreds of PV particles. Even [601_TD$IF]the [602_TD$IF]smaller exosomes, which are typically 100 nm in diameter, have the capacity to traffic multiple HAV or HEV viral particles [[603_TD$IF]64,81]. While this novel type of viral transmission was first reported with (+) ssRNA viruses [3], a giant DNA virus, Marseille virus, was recently discovered to also traffic between cells within micrometer vesicles, with each vesicle carrying hundreds of viral particles[14_TD$IF] [[604_TD$IF]98]. One of the implications of this novel [605_TD$IF]membrane [60_TD$IF]bound vesicle-mediated viral transmission is that it enables single cells to be infected simultaneously with multiple viral particles (Figure 2). [607_TD$IF]This type of viral transmission [608_TD$IF]also [609_TD$IF]yields greater infection efficiency and higher replication rates in a given population of cells compared with infection with similar numbers of free independent viral particles [[610_TD$IF]3,4,66,98]. For RNA viruses in general, collective travel and infection may be an important mechanism to overcome the drawbacks of errors during replication[61_TD$IF]: many viral RNA polymerases [612_TD$IF]lack proofreading [613_TD$IF]and [614_TD$IF]thus [615_TD$IF]end up synthesizing a heterogeneous populations of viral quasispecies [[61_TD$IF]99]. Even small genetic differences among quasispecies can manifest themselves,[17_TD$IF] in the next infection cycle, in differing rates of RNA translation, RNA synthesis,[105_TD$IF] particle assembly, or resistance to innate immune defenses[18_TD$IF] [[617_TD$IF]100,101]. While these mutations might [618_TD$IF]seal the replicative fate of [619_TD$IF]free [620_TD$IF]circulating [621_TD$IF]viral [62_TD$IF]particles, [623_TD$IF]by [624_TD$IF]traveling [142_TD$IF]and [625_TD$IF]infecting as[120_TD$IF] a population,[62_TD$IF] and through cooperative interactions among quasispecies, [54_TD$IF]such as sharing genetic templates, replication machinery, structural proteins, and innate immune defense modulators, [627_TD$IF]a greater overall [628_TD$IF]replication yield, resistance to antivirals and[30_TD$IF] possibly long-term fitness [629_TD$IF]may [630_TD$IF]be [631_TD$IF]achieved [[632_TD$IF]101] (Figure 2). Future studies will[63_TD$IF] be needed to fully test this hypothesis and also determine which types of viral cooperative interaction can enhance replicative fitness, what the relative contributions of cooperativity versus competition are, and whether vesicular travel of viral populations, compared with free viral particles, leads to greater genetic diversity over time. Finally, [634_TD$IF]these findings may [635_TD$IF]provide an opportunity to develop[63_TD$IF] entirely new [637_TD$IF]types of therapeutics that[12_TD$IF] by [638_TD$IF]attacking the cellular membrane trafficking pathways that enable populations of viruses to be captured and released (i.e., secretory autophagy or secretory MVBs) or by disrupting the integrity of [639_TD$IF]these membranes after release[640_TD$IF], [641_TD$IF]can [642_TD$IF]lead to inhibiting viral replication and drug resistance.

Concluding Remarks Recent investigations have revealed[49_TD$IF] novel ways by which membranes [643_TD$IF]integrate [64_TD$IF]themselves [645_TD$IF]into the viral life cycle: from facilitating viral genome synthesis to enabling viral populations to be transported out of cells and enhancing viral infectivity. Future studies will determine the extent of dependence on specific lipids among viruses, including PI4P, PE, PS, [64_TD$IF]among others, for replication and transmission; evaluate the long-term impact of manipulating lipid metabolism on cell physiology; and identify the machinery regulating secretory autophagy and secretory MVB pathways (see Outstanding Questions). Progress in these areas will lead to not only novel antiviral therapeutics, but also tools for manipulating these pathways in uninfected cells, in particular for those that regulate the production of extracellular vesicles, which are critical for cell

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Outstanding Questions How widespread [359_TD$IF]are PI4P[92_TD$IF]/[2_TD$IF]cholesterol and PE[92_TD$IF]/[2_TD$IF]cholesterol dependence among viruses? How does PI4P or PE docking regulate viral enzymatic activities? What is the long-term impact[650_TD$IF] on cell physiology of viral manipulation of lipid metabolism[123_TD$IF]? [651_TD$IF]What [652_TD$IF]impact [653_TD$IF]do [654_TD$IF]PS [65_TD$IF]lipid-enriched membranes surrounding exiting viruses [65_TD$IF]have [657_TD$IF]on the in vivo[125_TD$IF] immune response? What are the cellular and viral components regulating secretory autophagy and secretory MVB pathways? How are viral particles recognized by these pathways? How do the extracellular vesicles carrying viral particles transfer [658_TD$IF]their [659_TD$IF]cargo to the cells? What types of viral cooperative interactions enhance replicative fitness? What are the relative contributions of cooperativity versus competition? Does vesicular transmission of viral populations, compared to free viral particles, lead to greater genetic diversity?

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to cell communication and tissue remodeling. Finally, with the [647_TD$IF]constantly [648_TD$IF]evolving vast repertoire of cutting edge tools[649_TD$IF] available in microscopy, lipidomics, proteomics, and genetics, this is an exciting time to be studying viruses, which so often ends up being the study of cellular life. References 1.

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