viruses

Review Retrograde Motors and Their Role in Retroviral Transport

Gianfranco Pietrantoni, Rodrigo Ibarra-Karmy and Gloria Arriagada *

Instituto de Ciencias Biomedicas, Facultad de Medicina y Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago 8370071, Chile; [email protected] (G.P.); [email protected] (R.I.-K.) * Correspondence: [email protected]

 Received: 16 March 2020; Accepted: 22 April 2020; Published: 24 April 2020 

Abstract: Following entry into the host cell, generate a dsDNA copy of their genomes via reverse transcription, and this viral DNA is subsequently integrated into the chromosomal DNA of the host cell. Before integration can occur, however, retroviral DNA must be transported to the nucleus as part of a ‘preintegration complex’ (PIC). Transporting the PIC through the crowded environment of the cytoplasm is challenging, and retroviruses have evolved different mechanisms to accomplish this feat. Within a eukaryotic cell, act as the roads, while the microtubule-associated and kinesin are the vehicles that viruses exploit to achieve retrograde and anterograde trafficking. This review will examine the various mechanisms retroviruses have evolved in order to achieve retrograde trafficking, confirming that each has its own strategy to functionally subvert microtubule associated proteins.

Keywords: retroviruses; microtubules; dynein; traffic

1. Introduction As obligate intracellular parasites, viruses rely on cellular functions to replicate. A virus will make use of the microtubule network to transport and deliver their genome to the replication site inside a cell, a step that is crucial for the viral replication process, and particularly important for viruses that replicate in the nucleus, such as members of the retroviridae family. Limitations to free diffusion in the cytoplasm have forced viruses to develop efficient mechanisms to subvert the transport systems of the host. Through the use of different microtubule depolymerizing agents, namely, colchicine, vinblastine and nocodazole, it has been shown how relevant microtubule stability is for viral infection [1–3]. Many different viruses use the microtubule associated motor proteins to move along microtubules within the cell [1–5]. This review is focused on the mechanism retroviruses use to exploit and subjugate cellular machinery like the microtubule motor dynein for movement. Great advances have been made for HIV-1, but for other retroviruses there have been little advances.

2. Microtubules and Their Motor Proteins Microtubules are the road for the long-distance transport of endocytic/exocytic vesicles, organelles, and viral complexes [2,6,7]. This complex network of tubes is formed by heterodimers of α and β tubulin that interlace in a head-to-tail arrangement forming protofilaments, 13 protofilaments assemble to form a single microtubule filament. A key characteristic of these microtubules is their structural polarity, where one end of the microtubule considered a plus end, can quickly expand or shrink as required (dynamic instability), while the opposite minus end is attached to the microtubule organizing center (MTOC), allowing for directional growth of the microtubule network.

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The directional transport that occurs along the microtubules is accomplished by the actions of kinesins and (Table1), two ATP-dependent motor proteins. Kinesins are responsible for the VirusesViruses 2020 2020, 12, 12, x, xFOR FOR PEER PEER REVIEW REVIEW 2 2of of 11 11 transport of cargo within the cell in an anterograde manner, meaning, from the minus to the plus end of theTheThe microtubule directional directional transport [ 8transport,9], on thethat that otheroccurs occurs hand, along along thethe the cytoplasmicmicrotubules microtubules dyneinis is accomplished accomplished motors areby by the inthe chargeactions actions ofof of the retrogradekinesinskinesins transport and and dyneins dyneins that (Table occurs, (Table 1), 1), from two two theATP-dependent ATP-dependent plus end towards motor motor theproteins. proteins. MTOC Kinesins Kinesins or minus are are endresponsible responsible [10,11]. for Therefor the the are two kindstransporttransport of dynein,of of cargo cargo within dynein within the the 1 orcell cell cytoplasmic in in an an anterograd anterograd dynein,e emanner, manner, capable meaning, meaning, of mediating from from the the retrogrademinus minus to to the the transportplus plus end end of cargoofof in the the microtubule microtubule cytoplasm, [8,9], and[8,9], dyneinon on the the 2,other other also hand, knownhand, the the as cytoplasmic axonemalcytoplasmic dynein, dynein dynein responsiblemotors motors are are in forin charge thecharge transport of of the the in ciliaretrograde andretrograde flagella transport transport [7,10,12 that, 13that]. occurs, occurs, from from the the plus plus en endd towards towards the the MTOC MTOC or or minus minus end end [10,11]. [10,11]. There There areAare big two two part kinds kinds of of all of dynein, kinesinsdynein, dynein dynein transit 1 1or towardsor cytoplasmic cytoplasmic the plus dynein, dynein, end capable ofcapable the microtubule, of of mediating mediating nevertheless,retrograde retrograde transport transport there is a of cargo in the cytoplasm, and dynein 2, also known as axonemal dynein, responsible for the transport smallof number cargo in thatthe cytoplasm, moves in and the oppositedynein 2, also direction known [8 as,9 ,axonemal14]. The kinesindynein, responsible superfamily for is the composed transport of a in cilia and flagella [7,10,12,13]. widein variety cilia and of flagella different [7,10,12,13]. kinesins who vary in shape and form. An archetype of the kinesin family AA big big part part of of all all kinesins kinesins transit transit towards towards the the plus plus end end of of the the microtubule, microtubule, nevertheless, nevertheless, there there is is kinesin-1 also known as the “conventional” kinesin, forms heterotetramers composed of a pair of a asmall small number number that that moves moves in in the the opposite opposite direction direction [8,9,14]. [8,9,14]. The The kinesin kinesin superfamily superfamily is is composed composed of of heavy chains (HC) and a pair of light chains (LC). Each of the HC dimers links with two copies of the a awide wide variety variety of of different different kinesins kinesins who who vary vary in in sh shapeape and and form. form. An An archetype archetype of of the the kinesin kinesin family family LC dimer, it is worth noting that despite the fact that HC and LC dimers can be encoded by different kinesin-1kinesin-1 also also known known as as the the “conventional” “conventional” kinesi kinesin,n, forms forms heterotetramers heterotetramers composed composed of of a apair pair of of ,heavyheavy each chains chains kinesin-1 (HC) (HC) tetramer and and a apair pair is of comprisedof light light chains chains of (LC). two(LC). Ea identical Eachch of of the the HC HC HC and dimers dimers LC dimerslinks links with with [15 two]. two Cargo copies copies carried of of the the by kinesinsLCLC dimer, bindsdimer, it directlyit is is worth worth to noting noting specific that that sites despite despite located the the fact onfact thethat thatHC HC HC partand and LC of LC thedimers dimers kinesin-1 can can be be tetramer,encoded encoded by thisby different different cargo can interactgenes,genes, with each each the kinesin-1 motorkinesin-1 via tetramer tetramer tetratricopeptide is is comprised comprised repeats of of two two which identical identical are HC inHC thean and C-terminald LC LC dimers dimers end[15]. [15]. ofCargo Cargo the kinesincarried carried LC. Additionally,byby kinesins kinesins the binds binds expression directly directly to of to specific severalspecific sites LCsites kinesinlocated located on spliceon the the HC variantsHC part part of of that the the kinesin-1 possess kinesin-1 atetramer, tetramer, different this this C-terminal cargo cargo domain,cancan interact willinteract greatly with with contributethe the motor motor tovia via binding tetratricopeptide tetratricopeptide specificity repeats repeats of diff erentwhich whichtypes are are in in of the cargothe C-terminal C-terminal [9,14,16 end]. end of of the the kinesinThekinesin dynein LC. LC. Additionally, complexAdditionally, (Table the the1 expression) expression is a minus of of end-directed several several LC LC kinesin kinesin motor splice proteinsplice variants variants responsible that that possess possess for a widea adifferent different range of C-terminal domain, will greatly contribute to binding specificity of different types of cargo [9,14,16]. aspectsC-terminal of intracellular domain, motilitywill greatly including contribute retrograde to binding axonal specificity transport of different [17], organelle types of cargo transport [9,14,16]. [10, 18], The dynein complex (Table 1) is a minus end-directed motor responsible for a wide range and virusThe retrograde dynein complex transport (Table [1, 21)]. is This a minus complex, end-dire withcted amotor molecular protein mass responsible of 1.6MDa, for a wide is composed range of aspects of intracellular motility including retrograde axonal transport [17], organelle transport of twoof heavyaspects chains,of intracellular where several motility ATPase including domains retrograde are located,axonal transport two intermediate [17], organelle chains transport (IC), three [10,18],[10,18], and and virus virus retrograde retrograde transport transport [1,2]. [1,2]. This This complex, complex, with with a amolecular molecular mass mass of of 1.6MDa, 1.6MDa, is is types of light chains (LC) that bind to the IC at different spots, DYNLT, DYNLL and DYNLRB, two composedcomposed of of two two heavy heavy chains, chains, where where several several ATPase ATPase domains domains are are located, located, two two intermediate intermediate chains chains additional light intermediate chains (LIC) also bind to the DHC in an autonomous manner. Each of (IC),(IC), three three types types of of light light chains chains (LC) (LC) that that bind bind to to the the IC IC at at different different spots, spots, DYNLT, DYNLT, DYNLL DYNLL and and the DHCDYNLRB,DYNLRB, encompasses two two additional additional six ATPase light light intermediate intermediate units (AAA1-6) chains chains in (LIC) tandem,(LIC) also also allbind bind of to themto the the DHC organize DHC in in an an in autonomous aautonomous circular ring shape,manner.manner. that binds Each Each toof of the the DHC microtubulesDHC encompasses encompasses thanks six six ATPase toATPase a globular un unitsits (AAA1-6) domain(AAA1-6) thatin in tandem, tandem, forms all at all theof of them them end organize of organize the stalk. In vertebrateinin a acircular circular organisms, ring ring shape, shape, each that that of binds binds the dyneinto to the the microtubules microtubules ICs, LICs and thanks thanks LCs to areto a aglobular codifiedglobular domain bydomain two that genes,that forms forms dynein at at the the ICs and LICsendend of mayof the the alsostalk. stalk. be In In alternatively vertebrate vertebrate organisms, organisms, spliced and each each show of of the the di dyneinff dyneinerent phosphor-isoformsICs, ICs, LICs LICs and and LCs LCs are are [19 codified codified,20], the by by presence two two of thesegenes,genes, variations dynein dynein ICs ICs suggests and and LICs LICs that may may the also also di be ffbeerent alternatively alternatively dynein spliced versionsspliced and and are show show very different different likely phosphor-isoforms tophosphor-isoforms perform different tasks[19,20], by[19,20], binding the the presence presence preferentially of of these these tovari vari aations favoredations suggests suggests type of that that cargo. the the different Manydifferent studies dynein dynein have versions versions also are shownare very very thatlikely likely dynein to to requiresperformperform other different different molecules tasks tasks to by work by binding binding in an preferentially optimalpreferentially condition, to to a afavored favored these accessorytype type of of cargo. cargo. proteins Many Many which studies studies aff haveect have dynein’s also also shown that dynein requires other molecules to work in an optimal condition, these accessory proteins mechanochemistryshown that dynein such requires as dynactin, other molecules NudEL to/NudE, work in Lis1, an optimal and Bicaudal condition, D2, these among accessory others, proteins are also which affect dynein’s mechanochemistry such as dynactin, NudEL/NudE, Lis1, and Bicaudal D2, implicatedwhich affect to play dynein’s a role inmechanochemistry cargo binding and such processivity as dynactin, [21 NudEL/NudE,–27]. Lis1, and Bicaudal D2, amongamong others, others, are are also also implicated implicated to to play play a arole role in in cargo cargo binding binding and and processivity processivity [21–27]. [21–27]. Table 1. Motor proteins and their roles on viral infection. TableTable 1. 1. Motor Motor proteins proteins and and their their roles roles on on viral viral infection. infection.

StructureStructure CellularCellular Cellular Function Function Function Re Relevancelevance Relevance for for Viral Viral for ViralInfection Infection Infection DyneinDynein - Participates in retrograde organelle - Participates in retrograde organelle - Participates- Participates in virus retrograde in virus retrograde transport, - Participates in retrograde organelle transport- Participates in virus retrograde transport, transporttransport inside inside the the cell. cell. transport, carrying viral PIC towards inside the cell. carryingcarrying viral viral PIC PIC towards towards the the nucleus nucleus [23]. [23]. - -Plays Plays crucial crucial role role regulating regulating stages stages of of the nucleus [23]. - Plays crucial role regulating stages of cell cycle.- -Interacts Interacts with with HIV-1 HIV-1 by by associating associating with with cellcell cycle. cycle. - Interacts with HIV-1 by associating - Requires other molecules to work optimally BicD2BicD2 [28,29]. [28,29]. - -Requires Requires other other molecules molecules to to work work with BicD2 [28,29]. that also play a role in cargo binding - -Interacts Interacts directly directly with with BIV BIV capsid capsid for for optimallyoptimally that that also also play play a arole role in in cargo cargo - Interacts directly with BIV capsid for NudEL/NudE, Lis1, bicaudal D2). retrograderetrograde transport transport [19]. [19]. bindingbinding NudEL/NudE, NudEL/NudE, Lis1, Lis1, bicaudal bicaudal D2). D2). retrograde transport [19].

Dynactin DynactinDynactin - -Cofactor Cofactor that that activates activates dynein dynein mediates mediates - Cofactorvesicle that mobility. activates dynein mediates vesicle mobility. - Required- Required for retroviral for retroviral infection, infection, possibly - Links dyneinvesicle to various mobility. cargos. - Required for retroviral infection, possibly - Links dynein to various cargos. possibly mediating association to - Links dynein to various cargos. mediatingmediating association association to to dynein dynein [20,29]. [20,29]. - -Strengthens Strengthens dynein dynein binding binding to to dynein [20,29]. - Strengthens dynein binding to microtubules. microtubules.microtubules.

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Table 1. Cont.

VirusesStructure 2020, 12, x FOR PEER REVIEW Cellular Function Relevance for Viral Infection3 of 11

- Kinesin- Kinesin 4, 8 and 4, 8 and13 actively 13 actively regulate regulate KinesinKinesin microtubule dynamics, aiding in the microtubule dynamics, aiding in the process - Participates in anterograde transport process of infection [30]. - Participates in anterograde transport within of infection [30]. - Kinesin-1 adaptor, FEZ1 has been within thethe cell. cell. - Kinesin-1shown adaptor, to regulate FEZ1 has early been transport shown ofto - Contains- Contains a kinesin a kinesin “tail” “tail” where where cargo cargo binds regulateviral early particles transport by of associating viral particles to HIV-1 by binds andand a amotor motor domain domain responsible responsible for for its associating to HIV-1capsid capsid [31]. [31]. its movement.movement. - Kinesin -might Kinesin also might play alsoa role play in uncoating a role in of HIV-1uncoating thanks to ofFEZ1 HIV-1 and thanks Kif5B to[32,33]. FEZ1 and Kif5B [32,33]. 3. Retrovirus Replication 3. RetrovirusThe Replicationretroviral genome consists of two copies of a single stranded positive-sense RNA enclosed Thein a retroviral protein core, genome the genome consists contains of two copies three ofmain a single genes stranded gag, positive-senseand , that encode RNA enclosedthe viral in a proteinproteins. core, Retroviruses the genome enter contains cells threeby binding main to genes specificgag, cellular pol and receptorsenv, that on encode the plasma the viral membrane. proteins. RetrovirusesThen, the enter virion cells membrane by binding fuses to specificwith the cellularhost membrane receptors either on the at plasmathe cell membrane.surface or after Then, internalization into endosomes, as a result, the core containing the viral genome and viral enzymes, the virion membrane fuses with the host membrane either at the cell surface or after internalization is delivered into the cytoplasm. In the cytoplasm the core particle becomes the reverse transcription into endosomes, as a result, the core containing the viral genome and viral enzymes, is delivered into complex as it is exposed to nucleotide triphosphates, then reverse transcription (catalyzed by the the cytoplasm. In the cytoplasm [RT]) of thethe coreRNA particle genome becomesinto double-stranded the reverse transcriptionDNA occurs. The complex viral DNA as it is exposedmust to traffic nucleotide to the triphosphates,nucleus as part thenof the reverse pre-integration transcription complex (catalyzed (PIC). While by the the reversefull composition transcriptase of [RT]) ofthe the different RNA genome retroviral into PICs double-stranded is still unknown, DNA it is certain occurs. that The it viralmust DNAat least must contain traffi thec to viral the DNA, nucleus as partthe of the pre-integration (IN) and the complex capsid (PIC). (CA) Whileor Gag the proteins. full composition After gaining of the entry diff erentto the retroviralnucleus, IN PICs is stillpermanently unknown, itintegrates is certain the that viral it mustDNA atinto least the containhost genome, the viral after DNA,which thethe integraseintegrated (IN)provirus and is the capsidformed (CA) or[34]. Gag proteins. After gaining entry to the nucleus, IN permanently integrates the viral DNA intoThis the hostnewly genome, integrated after provirus which theis transcribed integrated in provirusto viral RNAs is formed by the [34 action]. of the host RNA Thispolymerase newly integratedII and subsequently provirus processed is transcribed and exported into viral to the RNAs cytoplasm. by the The action exported of the RNA host has RNA polymerasetwo possible II and subsequentlyfates: it can be processed taken to and ribosome exporteds and to thetranslated cytoplasm. to produce The exported the viral RNA proteins. has two Alternatively, it can be used to compose the genome of new viral particles. After a virus is fully possible fates: it can be taken to ribosomes and translated to produce the viral proteins. Alternatively, assembled within the infected cell, it travels to the cell membrane where it buds out, as an immature it can be used to compose the genome of new viral particles. After a virus is fully assembled within the particle. The maturation occurs in a process triggered by the viral protease [34], where the protease infectedcleaves cell, itGag travels and Gag-Pol to the cell to membraneform the virion. where Each it buds phase out, in asthis an elaborate immature mu particle.lti-stage Theprocess, maturation from occursentry in a to process budding, triggered not only by requires the viral the proteaseaid of multip [34],le wherehost proteins the protease but also cleaves the cytoskeleton. Gag and In Gag-Pol fact, to formfrom the an virion. infectious Each retrovirus phase in thisstandpoint, elaborate the multi-stage cytoskeleton process, of the host from cell entry acts to as budding, a pathway not that only requiresallows the aidthe ofintracellular multiple hostmovement proteins required but also by the a retrovirus cytoskeleton. for a Insuccessful fact, from infection. an infectious retrovirus standpoint, the cytoskeleton of the host cell acts as a pathway that allows the intracellular movement required4. HIV-1 by a retrovirusand Microtubule for a successful Associated infection. Proteins at Early Infection Retroviruses depend upon microtubule associated dynein for their retrograde transport inside 4. HIV-1 and Microtubule Associated Proteins at Early Infection the host cell, were each retrovirus have a particular dynein partner to mediate the movement Retroviruses[19,21,28,29,35,36]. depend In the upon last 5 microtubuleyears, most research associated attention dynein has focused for their on retrogradeHIV-1, the causative transport insideagent the of host AIDS, cell, and were less advance each retrovirus has been made have re agarding particular other dyneinretroviruses partner and their to mediateassociation the movementwith the[19 ,21cytoskeleton.,28,29,35,36 ]Thus,. In thewe lastwill 5first years, pres mostent the research history attention of research has into focused and oncurrent HIV-1, understanding of how HIV-1 subverts microtubule associated proteins for efficient retrograde the causative agent of AIDS, and less advance has been made regarding other retroviruses and their transport to the cell nucleus. association with the cytoskeleton. Thus, we will first present the history of research into and current For HIV-1, the first demonstration that retrograde transport of the PIC occurs along understanding of how HIV-1 subverts microtubule associated proteins for efficient retrograde transport microtubules and utilizes dynein comes from the Hope laboratory [23]. This group developed a to thesystem cell nucleus. that is now widely used to study the intracellular transport of HIV-1: a virion that contains Forfluorescent HIV-1, the first protein demonstration and can label that the retrograde incoming transportparticle, and of thea fluorescent PIC occurs protein along fused microtubules to the and utilizesfirst 15 amino dynein acids comes of Scr from (S15-fluorescent the Hope laboratory protein), that [23 is]. used This as groupa marker developed of unfused a virions. system Using that is now widelythese tools, used coupled to study with the live intracellular imaging, they transport showed of that HIV-1: when aanti-IC virion that contains were fluorescent microinjected Vpr proteinto andcells,can viral label movement the incoming and transport particle, towards and ath fluorescente nucleus was protein halted, fused but only to theduring first the 15 early amino acids ofstages Scr (S15-fluorescentof infection [23]. protein),This raised that a is question used as: aWhat marker cellular of unfused partner virions. mediates Using this thesedirected tools, coupledmovement? with live imaging, they showed that when anti-IC antibodies were microinjected to cells,

Viruses 2020, 12, 483 4 of 11 viral movement and transport towards the nucleus was halted, but only during the early stages of infection [23]. This raised a question: What cellular partner mediates this directed movement? In the search for the dynein partner of HIV-1 it was shown that infection can be inhibited by using a dynein light chain peptide belonging to the 19 amino acids of the DYNLL C-terminal [37], but DYNLL did not progress as a relevant factor for HIV-1 infection, although it is essential for other retroviruses (see below). Later, Gallo and Hope reported that the light chain of axonemal dynein, DNAL1, was required for effective HIV-1 infection regardless of viral entry. They also reported that (MLV) depends on DNAL1 for infection, though not as prominently as HIV-1 [38]. While a newly arrived HIV-1 particle presents a retrograde net movement, live cell imaging has shown displays of bi-directional movement on microtubules [23,31,39]. In fact, FEZ1, the kinesin-1 heavy chain adaptor, regulates the early transport of incoming viral particles by associating to the HIV-1 capsid [31,40]. This confirmed the tug-of-war model, wherein both anterograde and retrograde transport of cargo takes place, although the net movement is retrograde. When treating cells with nocodazole, an antineoplastic agent that disrupts dynamic microtubule polymerization, most microtubules will be affected and a small number of microtubules, the stable microtubules, will remain intact. When testing HIV-1 infection after treating cells with nocodazole, infection was not completely abolished [23], this suggested that HIV-1 might use microtubule dependent and independent trafficking for infection, or that HIV-1 was capable of enhancing microtubule stabilization in order to aid its infection process. Microtubule stabilization upon HIV-1 infection was demonstrated by the Nhagavi laboratory [41]. All microtubules contain an end binding protein called EB1 which acts as a stabilizing cap at the end of each microtubule, the Nhagavi laboratory showed that by depleting EB1, the number of stable microtubules and consequently, HIV-1 rate during early infection steps was reduced [41]. They also showed that the EB1-binding protein, Kif4, is targeted by the HIV-1 matrix protein (MA) to induce the stabilization of microtubules through the recruitment of EB-1 to the plus tip of the microtubule [41–43]. EB1 recruits other +Tips to stabilized microtubules, among them are the diaphanous related-proteins Dia1 and Dia2. Use of siRNA has shown that both Dia1 and Dia2 coordinate microtubule stability and capsid disassembly, thus regulating uncoating. Silencing of Dia1 or Dia2 reduces HIV-1 infection regardless of the entry mechanism and the cell type; formation of stable microtubules upon HIV-1 infection was also lost, showing that the HIV-1 mechanism of microtubule stabilization is even more complex than previously described [44]. Another protein that participates in microtubule stabilization is microtubule-associated protein 1 (MAP1) [45]. Although not a motor protein, MAP1 has a role in the regulation and cargo-binding of microtubule associated motors [46]. The Arhel laboratory showed that MAP1 can directly interact with HIV-1 p24 capsid protein. Depletion of MAP1 reduced capsid cores association to both stable and dynamic microtubules; the authors suggest that MAP1 might tether capsid cores to the microtubule network, thus promoting cytoplasmic trafficking [47]. These findings confirmed the need of stable microtubules for HIV-1 infection and suggest that MAP1 might provide an anchor point for HIV-1, mediated by p24 at the plus end of the microtubules, before trafficking using dynein activity with an (at that time) unknown partner mediating the association. It took some time to find a proper partner to pair up HIV-1 PIC with dynein. Two independent articles using different experimental approaches showed that BicaudalD2 (BicD2) was the missing link. BicD2 is a multi-purpose adaptor of dynein that plays a crucial role on development [48–50] organelle trafficking [51–53] and nuclear positioning; it has also been reported to have important roles in microtubule organization and lipid droplet transport [48,54,55]. It has been suggested that BicD2 functions as a modular link between dynein and the cargo; the N-terminal coiled coil of BicD2 interacts with dynein, whereas the C-terminal coiled coil binds to cargo-specific factors. BicD2 was initially identified as a cellular co-factor for HIV-1 infection in one [56] of the 3 genome-wide screens developed using either shRNA or siRNA [56–58] published in 2008. It was not immediately studied, since other proteins seemed to be more attractive at that time for those following the mechanism of the hits described in those screens. Viruses 2020, 12, 483 5 of 11

For a while, the research community wondered if indeed a direct interaction with the dynein machinery was occurring, and many, including us, were looking only at dynein and dynactin core components, without much success. In 2017, the Campbell laboratory showed that BicD2 is an essential host factor required for infection [28]. For this, they performed a CRISPR/Cas9 knock out of BicD2 in different cellular models. They showed that BicD2 interacts with the incoming particle and is required for the trafficking of fused particles to the nucleus and for the nuclear import of the viral genome. The direct interaction of BicD2 and CA was shown in vitro using the CA-NC assemblies binding assay and confirmed in infected cells by proximity ligation assay (PLA) [28]. Moreover, they have shown that the KO of BicD2 leads to the expression of interferon stimulated (ISG) upon HIV-1 infection, suggesting that in the absence of BicD2, viral DNA accumulates in the cytosol leading to an antiviral response, while in presence of BicD2, the PIC will travel rapidly towards the nucleus along microtubules, and avoid detection by the cellular innate immune machinery. The Aiken laboratory reported a similar finding in early 2018 [29]. Using a different strategy, silencing by siRNAS, they demonstrated that not only BicD2, but also dynactin backbone complex (ACTR1 and DCTN2/DCTN3) facilitate the transport and infection of HIV-1, confirming that BicD2 is the adaptor between HIV-1 PIC and dynein. They also showed that BicD2 is also essential for simian immunodeficiency virus (SIV). Performing biochemical experiments, they delineate the domains of BicD2 that interact with HIV-1 CA. These two studies have finally provided the missing piece of the puzzle: which protein mediated the association of HIV-1 to dynein after the induction of stable microtubules. Now we can envision a model in which, after fusion, MA will recruit EB1 to induce stable microtubules helped by other +Tips, MAP1 will anchor the core to the microtubules and, using BicD2 to piggyback, the core will be transported by dynein to the minus end of microtubules and from there the viral DNA will enter the nucleus (Figure1). We have described the process that yield a net movement to the minus end of the microtubules. But as mentioned above, there is indeed a bidirectional movement of the incoming HIV-1 capsid cores, mediated by kinesins. This bi-directional movement is also important for uncoating (loss of capsid). Uncoating is a highly regulated process necessary for nuclear entry, no uncoating or accelerated uncoating are deleterious for infection [32,44,59–61]. Although it is not clear how the mature capsid core disassembles to allow the nuclear translocation of the lentiviral genomes, two models have been debated. The first one proposed that capsid remains fully intact until it arrives to the nuclear pore, and its disassembly occurs as the viral DNA is imported into the nucleus. The second model proposes that capsid undergoes structural changes in the cytoplasm resulting in capsid rupture where a portion of capsid remains intact until disassembly is completed at the nuclear pore, this model agrees with the idea that during trafficking along microtubules, cytoplasmic dynein and kinesin 1, particularly Kif5B, are required for this process as the genetic inhibition of DHC and Kif5B by siRNA, or pharmacological inhibition of dynein function, delayed uncoating, and reduced HIV-1 infection [33]. Furthermore, FEZ1 a kinesin adaptor that bridge HIV-1 cores to Kif5B is important for both traffic and uncoating [31–33], were phosphorylation of FEZ1 is required to recruit Kif5B to the HIV-1 cores and to mediate uncoating along movement [32]. If FEZ1 and Kif1B promote uncoating and BicD2 protect from accelerated uncoating and/or rapidly move HIV-1 PIC to the nuclear pore area [28], there must be a highly regulated balance that couples trafficking with uncoating to allow sufficient loss of capsid to permit nuclear entry, but a such a rate that viral DNA is not detected by the antiviral machinery in the cytoplasm. Which other players are regulating this process is still a matter of debate. Viruses 2020, 12, 483 6 of 11

Viruses 2020, 12, x FOR PEER REVIEW 6 of 11

FigureFigure 1. Directed 1. Directed retrograde retrograde transport transport ofof HIV-1HIV-1 alongalong microtubules. Directed Directed movement movement along along microtubules is carried out by the ATP-dependent complexes kinesin and dynein, were kinesins are microtubules is carried out by the ATP-dependent complexes kinesin and dynein, were kinesins are the anterograde motor and dynein is the retrograde motor. This transport can occur along stable and the anterograde motor and dynein is the retrograde motor. This transport can occur along stable and dynamic microtubules. While both kinesin and dynein play crucial roles during retroviral infection, dynamicin this microtubules. figure, we focus While on the both role kinesin of cytoplasmi and dyneinc dynein, play leaving crucial kinesins roles duringanterograde retroviral transport infection, out, in this figure,as it plays we focusa bigger on role the during role of the cytoplasmic late stages dynein,of the infectious leaving cycle kinesins and release anterograde of new transport viral particles out, as it plays(1 a). bigger Once a rolesusceptible during cell the is late infected stages by ofHIV-1, theinfectious fusion of the cycle viral and andrelease plasma ofmembrane new viral will particles occur. (1). OnceThen, a susceptible the viral core cell and is infected the matrix by protein HIV-1, (MA) fusion are of released the viral into and the plasma cytoplasm, membrane MA by its will interaction occur. Then, the viralwith core Kif4 andwill therecruit matrix the end protein binding (MA) 1 prot areein released (EB1) to into the theplus cytoplasm, end of microtubules MA by its (2). interaction This event with Kif4 willwill allow recruit the the stabilization end binding of microtubules 1 protein (EB1)and othe tor the +Tip, plus such end as diaphanous-related of microtubules ( 2formins). This (DRF), event will allowcan the be stabilization recruited. Microtubule of microtubules associated and other proteins,+Tip, such such asas MAP1, diaphanous-related can be recruited formins in an HIV-1- (DRF), can be recruited.independent Microtubule manner ( associated3). HIV-1 core, proteins, through such the as capsid MAP1, protein can be (CA), recruited binds in to an the HIV-1-independent microtubule stabilization is microtubule-associated protein 1 (MAP1) and Bicaudal D2 (BicD2). It is possible to manner (3). HIV-1 core, through the capsid protein (CA), binds to the microtubule stabilization is speculate that MAP1 acts as a hook to attract HIV-1 cores to the microtubules and to the vicinity of microtubule-associated protein 1 (MAP1) and Bicaudal D2 (BicD2). It is possible to speculate that dynein (4). BicD2 acts as the bridge between HIV-1 core and dynein. A functional HIV-1 transport MAP1 acts as a hook to attract HIV-1 cores to the microtubules and to the vicinity of dynein (4). BicD2 complex is complete once it contains dynein, dynactin and BicD2 together. Then, a directed retrograde acts asmovement the bridge towards between the HIV-1microtubule core and organizing dynein. center A functional (MTOC) HIV-1will occur. transport Along complex the movement, is complete once it contains dynein, dynactin and BicD2 together. Then, a directed retrograde movement towards the microtubule organizing center (MTOC) will occur. Along the movement, reverse transcription and uncoating are simultaneously happening (5). Several cellular factors will help along the way to finally cross the nuclear pore and reach the nucleus. Viruses 2020, 12, 483 7 of 11

5. BIV, MLV and PFV directly Associate to Dynein Light Chains Less information is available for other retroviruses, even , which are mostly used for comparison, rather than objects of study. For example, bovine immunodeficiency virus (BIV), requires microtubules and dynein for retrograde transport. The capsid protein (CA) of BIV directly associates to dynein LC DYNLL [19], but it is not known whether BIV can induce microtubule stabilization or if any other component of the retrograde transport machinery or microtubule associated protein is required for infection. Primate foamy virus (PFV) is a virus from the spumavirus genus and the prototypic model for foamy virus replication. This virus also makes use of the cellular motor proteins, before nuclear entry, it first traffics to the microtubule organization center (MTOC) in an event requiring the activity of dynein. This was demonstrated by abolishing dynein activity within cells via overexpressing a dominant negative form of the dynactin component p150Glued. The association that occurs between dynein and PFV is triggered by the interaction between the dynein LC DYNLL and Gag [18], the association between the two was demonstrated by co-localization and co-immunoprecipitation assays. These two examples might lead one to think that unlike HIV-1 all other retroviruses associate to dynein by the LC DYNLL, but results of our laboratory tests showed something different [20,21]. Murine leukemia virus (MLV) is one of the most widely used virus models and is also used as a gene vector (both in vitro and in vivo). We have previously demonstrated that the dynein regulator p50/dynamitin, together with the dynein IC, associate with the MLV PIC [20], this led us to discover that dynein regulators p50 and NudEL are essential for MLV infection [20,29]. Dynein IC also plays a crucial role in MLV infection, since the silencing of the IC by shRNA reduced MLV infection, albeit not as strikingly as p50 or NudEL knock-downs [20]. Dynein IC is important for associating dynein with cargo and is essential to anchor LCs to the complex. The latter finding led us to study the role of LCs on MLV infection. Our research demonstrated that silencing of the LC DYNLRB2 reduced MLV infection. On the other hand, over-expression of the DYNLRB2 levels had an enhancing effect over MLV infection, showing a functional role for DYNLRB2 on the MLV infection process [21]. Even though there has been a lack of evidence for a direct physical interaction between the MLV proteins and DYNLRB2 and a clear role on MLV traffic along microtubules, tools such as fluorescent label virions were p12 is tagged with GFP [22] will allow us to further confirm the essential role of dynein and its LC DYNLRB2 in MLV early infection. Unpublished work from our group, using GFP-p12-labeled MLV, point to DYNLRB2 being essential for both trafficking MLV along the microtubules and its arrival to the nucleus (manuscript under preparation). Several HIV-1-focused reports that used MLV as a control or comparison have shown the requirement for dynein and dynactin activity, but do not agree on the importance of BicD2, which may be dispensable [28] or somehow required [29]. We can conclude that for MLV, BIV and PFV, dynein LC are important for retrograde movement, but direct or functional associations to dynein are mediated by specific LC (Figure2), and can speculate that if the binding of a retroviral core to dynein is direct, then the induction of stable microtubules is not necessary for infection, since MLV does not induce microtubule stabilization upon infection [41]. Viruses 2020, 12, x FOR PEER REVIEW 8 of 11

and DYNLRB2 and a clear role on MLV traffic along microtubules, tools such as fluorescent label virions were p12 is tagged with GFP [22] will allow us to further confirm the essential role of dynein and its LC DYNLRB2 in MLV early infection. Unpublished work from our group, using GFP-p12- labeled MLV, point to DYNLRB2 being essential for both trafficking MLV along the microtubules and its arrival to the nucleus (manuscript under preparation). Several HIV-1-focused reports that used MLV as a control or comparison have shown the requirement for dynein and dynactin activity, but do not agree on the importance of BicD2, which may be dispensable [28] or somehow required [29]. We can conclude that for MLV, BIV and PFV, dynein LC are important for retrograde movement, but direct or functional associations to dynein are mediated by specific LC (Figure 2), and can speculate that if the binding of a retroviral core to Virusesdynein2020 is ,direct,12, 483 then the induction of stable microtubules is not necessary for infection, since 8MLV of 11 does not induce microtubule stabilization upon infection [41].

Figure 2.2. RetrovirusesRetroviruses associate associate with with dynein dynein by directby direct interaction interaction with with light chains.light chains. Dynein Dynein light chains light arechains essential are essential for primate for foamy primate virus foamy (PFV), murinevirus (P leukemiaFV), murine virus leukemia (MLV) and virus Bovine (MLV) immunodeficiency and Bovine virusimmunodeficiency (BIV) infection. virus PFV and(BIV) BIV infection. directly associatePFV and through BIV directly Gag or associate CA to DYNLL through for theirGag retrogradeor CA to transport.DYNLL for MLV their requires retrograde dynein transport. and dynactin MLV requires for infection, dynein andand althoughdynactin nofor physicalinfection, interaction and although has beenno physical demonstrated, interaction the has light been chain demonstrated, DYNLRB2 is the functionally light chain required DYNLRB2 for MLVis functionally infection. required for MLV infection. 6. Conclusions 6. ConclusionsRetroviruses make use of the cellular machinery to achieve effective infection, and the microtubule cytoskeleton,Retroviruses as well make as the use dynein of the and cellular kinesin machinery motors, are to cellular achieve components effective thatinfection, viruses and require the andmicrotubule are used cytoskeleton, in many steps as of well a virus as the replication dynein cycle.and kinesin Though motors, the requirement are cellular for components this machinery that duringviruses therequire early and events are ofused infection in many is sharedsteps of among a virus the replication retroviruses, cycle. each Though virus appearsthe requirement to use these for componentsthis machinery in a during different the way. early Whereas events HIV-1 of infect subvertsion is the shared retrograde among transport the retroviruses, machinery byeach binding virus BicD2appears for to a use piggyback these components ride on dynein, in a different other retroviruses way. Whereas directly HIV-1 associate subverts todynein the retrograde components. transport It is possiblemachinery to speculateby binding that BicD2 these for mechanistic a piggyback di ridefferences on dynein, are related other to retroviruses the virus capacity directly to associate evade the to antiviraldynein components. machinery on It theis possible cell; MLV, to forspeculate example, that does these not mechanistic induce anantiviral differences state are and related alsoretains to the morevirus CAcapacity protein to associatedevade the antiviral to the preintegration machinery on complex the cell; than MLV, HIV-1, for example, since MLV does does not not induce have thean needantiviral to undergo state and uncoating also retains to fitmore through CA protein the nuclear associated pore, theto the structure preintegration of the capsid complex shell than might HIV- be used1, since to bindMLV directly does not to dynein have the components. need to underg HIV-1,o on uncoating the other hand,to fit mustthroug goh throughthe nuclear uncoating, pore, butthe instructure a very regulatedof the capsid manner, shell inmight order be to used evade to bind an antiviral directly response to dynein from components. the host. Thus,HIV-1, while on the moving other alonghand, themust microtubules, go through theuncoating, same proteins but in thata very are regulated subverting manner, for traffi incking order (BicD2 to evade and FEZ1an antiviral/Kif5B) areresponse helping from control the thehost. loss Thus, of capsid. while Moremoving research along isthe required microtubules, to confirm the if same this hypothesisproteins that is true, are andsubverting the study for of trafficking other retroviruses (BicD2 mustand FEZ1/Kif5B) be included. are helping control the loss of capsid. More One can also speculate that older retroviruses were directly interacting with the cellular motors, and as they were evolving, a diversification of the mechanisms with which to accomplish retrograde movement appeared. More research is needed to determine the cytoplasmic transport strategies of other retroviruses, since knowledge of how different retroviruses interact with the cytoskeleton will likely provide important new insights into their biology and evolution.

Funding: This research was funded by Fondo Nacional de Desarrollo Científico y Tecnológico grant FONDECYT1130852 and FONDECYT1180705 to GA. Acknowledgments: The authors would like to recognize all the researches that have worked to elucidate the mechanisms by which retroviruses subverts the microtubule-associated traffic machinery and apologized to all of those that were not mentioned in this review. Conflicts of Interest: The authors declare no conflict of interest. Viruses 2020, 12, 483 9 of 11

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