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Due to its central role in cell biology, the cytoskeleton is a key regulator of viral infection, influencing nearly every step of the viral life cycle. In this review, we will discuss the role of two key components of the cytoskeleton, namely the actin and microtubule networks in early HIV-1 infection. We will discuss key contributions to processes ranging from the attachment and entry of viral particles at the cell surface to their arrival and import into the nucleus and identify areas where further research into this complex relationship may yield new insights into HIV-1 pathogenesis., (© 2022 Federation of European Biochemical Societies.)

HIV-1 replication in macrophages and microglia involves intracellular assembly and budding into modified subsets of multivesicular bodies (MVBs), which support both viral persistence and spread. However, the cellular factors that regulate HIV-1's vesicular replication remain poorly understood. Recently, amyloid precursor protein (APP) was identified as an inhibitor of HIV-1 replication in macrophages and microglia via an unknown mechanism. Here, we show that entry of HIV-1 Gag into MVBs is blocked by the amyloidogenic C-terminal fragment of APP, "C99", but not by the non-amyloidogenic product, "C83". To counter this, Gag promotes multi-site ubiquitination of C99 which controls both exocytic sorting of MVBs and further processing of C99 into toxic amyloids. Processing of C99, entry of Gag into MVBs and release of infectious virus could be suppressed by expressing ubiquitination-defective C99 or by γ-secretase inhibitor treatment, suggesting that APP's amyloidogenic pathway functions to sense and suppress HIV-1 replication in macrophages and microglia., (© 2023. The Author(s).)

Fasciculation and elongation factor zeta 1 (FEZ1), a multifunctional kinesin-1 adaptor, binds human immunodeficiency virus type 1 (HIV-1) capsids and is required for efficient translocation of virus particles to the nucleus to initiate infection. However, we recently found that FEZ1 also acts as a negative regulator of interferon (IFN) production and interferon-stimulated gene (ISG) expression in primary fibroblasts and human immortalized microglial cell line clone 3 (CHME3) microglia, a natural target cell type for HIV-1 infection. This raises the question of whether depleting FEZ1 negatively affects early HIV-1 infection through effects on virus trafficking or IFN induction or both. Here, we address this by comparing the effects of FEZ1 depletion or IFN-β treatment on early stages of HIV-1 infection in different cell systems with various IFN-β responsiveness. In either CHME3 microglia or HEK293A cells, depletion of FEZ1 reduced the accumulation of fused HIV-1 particles around the nucleus and suppressed infection. In contrast, various doses of IFN-β had little to no effect on HIV-1 fusion or the translocation of fused viral particles to the nucleus in either cell type. Moreover, the potency of IFN-β's effects on infection in each cell type reflected the level of induction of MxB, an ISG that blocks subsequent stages of HIV-1 nuclear import. Collectively, our findings demonstrate that loss of FEZ1 function impacts infection through its roles in two independent processes, as a direct regulator of HIV-1 particle transport and as a regulator of ISG expression. IMPORTANCE As a hub protein, fasciculation and elongation factor zeta 1 (FEZ1) interacts with a range of other proteins involved in various biological processes, acting as an adaptor for the microtubule (MT) motor kinesin-1 to mediate outward transport of intracellular cargoes, including viruses. Indeed, incoming HIV-1 capsids bind to FEZ1 to regulate the balance of inward/outward motor activity to ensure net forward movement toward the nucleus to initiate infection. However, we recently showed that FEZ1 depletion also induces interferon (IFN) production and interferon-stimulated gene (ISG) expression. As such, it remains unknown whether modulating FEZ1 activity affects HIV-1 infection through its ability to regulate ISG expression or whether FEZ1 functions directly, or both. Using distinct cell systems that separate the effects of IFN and FEZ1 depletion, here we demonstrate that the kinesin adaptor FEZ1 regulates HIV-1 translocation to the nucleus independently of its effects on IFN production and ISG expression., Competing Interests: The authors declare no conflict of interest.

While HIV-1 infection of macrophages plays a major role in viral persistence and pathogenesis, how HIV-1 transfers from infected T cells to macrophages remains elusive. In this issue, Mascarau et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205103) demonstrate how macrophage polarization drives their ability to fuse with HIV-1 infected T cells via the CD81/RhoA-ROCK/Myosin axis., (© 2023 Naghavi.)

Microtubules (MTs) form rapidly adaptable, complex intracellular networks of filaments that not only provide structural support, but also form the tracks along which motors traffic macromolecular cargos to specific sub-cellular sites. These dynamic arrays play a central role in regulating various cellular processes including cell shape and motility as well as cell division and polarization. Given their complex organization and functional importance, MT arrays are carefully controlled by many highly specialized proteins that regulate the nucleation of MT filaments at distinct sites, their dynamic growth and stability, and their engagement with other subcellular structures and cargoes destined for transport. This review focuses on recent advances in our understanding of how MTs and their regulatory proteins function, including their active targeting and exploitation, during infection by viruses that utilize a wide variety of replication strategies that occur within different cellular sub-compartments or regions of the cell., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Fasciculation and elongation protein zeta-1 (FEZ1) is a multifunctional kinesin adaptor involved in processes ranging from neurodegeneration to retrovirus and polyomavirus infection. Here, we show that, although modulating FEZ1 expression also impacts infection by large DNA viruses in human microglia, macrophages, and fibroblasts, this broad antiviral phenotype is associated with the pre-induction of interferon-stimulated genes (ISGs) in a STING-independent manner. We further reveal that S58, a key phosphorylation site in FEZ1's kinesin regulatory domain, controls both binding to, and the nuclear-cytoplasmic localization of, heat shock protein 8 (HSPA8), as well as ISG expression. FEZ1- and HSPA8-induced changes in ISG expression further involved changes in DNA-dependent protein kinase (DNA-PK) accumulation in the nucleus. Moreover, phosphorylation of endogenous FEZ1 at S58 was reduced and HSPA8 and DNA-PK translocated to the nucleus in cells stimulated with DNA, suggesting that FEZ1 is a regulatory component of the recently identified HSPA8/DNA-PK innate immune pathway., Competing Interests: Declaration of interests The authors declare conflicting interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many viruses directly engage and require the dynein-dynactin motor-adaptor complex in order to transport along microtubules (MTs) to the nucleus and initiate infection. HIV type 1 (HIV-1) exploits dynein, the dynein adaptor BICD2, and core dynactin subunits but unlike several other viruses, does not require dynactin-1 (DCTN1). The underlying reason for HIV-1's variant dynein engagement strategy and independence from DCTN1 remains unknown. Here, we reveal that DCTN1 actually inhibits early HIV-1 infection by interfering with the ability of viral cores to interact with critical host cofactors. Specifically, DCTN1 competes for binding to HIV-1 particles with cytoplasmic linker protein 170 (CLIP170), one of several MT plus-end tracking proteins (+TIPs) that regulate the stability of viral cores after entry into the cell. Outside of its function as a dynactin subunit, DCTN1 also functions as a +TIP that we find sequesters CLIP170 from incoming particles. Deletion of the Zinc knuckle (Zn) domain in CLIP170 that mediates its interactions with several proteins, including DCTN1, increased CLIP170 binding to virus particles but failed to promote infection, further suggesting that DCTN1 blocks a critical proviral function of CLIP170 mediated by its Zn domain. Our findings suggest that the unique manner in which HIV-1 binds and exploits +TIPs to regulate particle stability leaves them vulnerable to the negative effects of DCTN1 on +TIP availability and function, which may in turn have driven HIV-1 to evolve away from DCTN1 in favor of BICD2-based engagement of dynein during early infection., Competing Interests: The authors declare no competing interest.

Microtubules (MTs) form a filamentous array that provide both structural support and a coordinated system for the movement and organization of macromolecular cargos within the cell. As such, they play a critical role in regulating a wide range of cellular processes, from cell shape and motility to cell polarization and division. The array is radial with filament minus-ends anchored at perinuclear MT-organizing centers and filament plus-ends continuously growing and shrinking to explore and adapt to the intracellular environment. In response to environmental cues, a small subset of these highly dynamic MTs can become stabilized, acquire post-translational modifications and act as specialized tracks for cargo trafficking. MT dynamics and stability are regulated by a subset of highly specialized MT plus-end tracking proteins, known as +TIPs. Central to this is the end-binding (EB) family of proteins which specifically recognize and track growing MT plus-ends to both regulate MT polymerization directly and to mediate the accumulation of a diverse array of other +TIPs at MT ends. Moreover, interaction of EB1 and +TIPs with actin-MT cross-linking factors coordinate changes in actin and MT dynamics at the cell periphery, as well as during the transition of cargos from one network to the other. The inherent structural polarity of MTs is sensed by specialized motor proteins. In general, dynein directs trafficking of cargos towards the minus-end while most kinesins direct movement toward the plus-end. As a pathogenic cargo, HIV-1 uses the actin cytoskeleton for short-range transport most frequently at the cell periphery during entry before transiting to MTs for long-range transport to reach the nucleus. While the fundamental importance of MT networks to HIV-1 replication has long been known, recent work has begun to reveal the underlying mechanistic details by which HIV-1 engages MTs after entry into the cell. This includes mimicry of EB1 by capsid (CA) and adaptor-mediated engagement of dynein and kinesin motors to elegantly coordinate early steps in infection that include MT stabilization, uncoating (conical CA disassembly) and virus transport toward the nucleus. This review discusses recent advances in our understanding of how MT regulators and their associated motors are exploited by incoming HIV-1 capsid during early stages of infection.

While the microtubule end-binding protein, EB1 facilitates early stages of HIV-1 infection, how it does so remains unclear. Here, we show that beyond its effects on microtubule acetylation, EB1 also indirectly contributes to infection by delivering the plus-end tracking protein (+TIP), cytoplasmic linker protein 170 (CLIP170) to the cell periphery. CLIP170 bound to intact HIV-1 cores or in vitro assembled capsid-nucleocapsid complexes, while EB1 did not. Moreover, unlike EB1 and several other +TIPs, CLIP170 enhanced infection independently of effects on microtubule acetylation. Capsid mutants and imaging revealed that CLIP170 bound HIV-1 cores in a manner distinct from currently known capsid cofactors, influenced by pentamer composition or curvature. Structural analyses revealed an EB-like +TIP-binding motif within the capsid major homology region (MHR) that binds SxIP motifs found in several +TIPs, and variability across this MHR sequence correlated with the extent to which different retroviruses engage CLIP170 to facilitate infection. Our findings provide mechanistic insights into the complex roles of +TIPs in mediating early stages of retroviral infection, and reveal divergent capsid-based EB1 mimicry across retroviral species., (© 2020 The Authors.)

Human immunodeficiency virus type 1 (HIV-1) exploits a number of specialized microtubule (MT) plus-end tracking proteins (commonly known as +TIPs) to induce the formation of stable microtubules soon after virus entry and promote early stages of infection. However, given their functional diversity, the nature of the +TIPs involved and how they facilitate HIV-1 infection remains poorly understood. Here, we identify cytoplasmic linker-associated protein 2 (CLASP2), a +TIP that captures cortical MT plus ends to enable filament stabilization, as a host factor that enables HIV-1 to induce MT stabilization and promote early infection in natural target cell types. Using fixed- and live-cell imaging in human microglia cells, we further show that CLASP2 is required for the trafficking of incoming HIV-1 particles carrying wild-type (WT) envelope. Moreover, both WT CLASP2 and a CLASP2 mutant lacking its C-terminal domain, which mediates its interaction with several host effector proteins, bind to intact HIV-1 cores or in vitro -assembled capsid-nucleocapsid (CA-NC) complexes. However, unlike WT CLASP2, the CLASP2 C-terminal mutant is unable to induce MT stabilization or promote early HIV-1 infection. Our findings identify CLASP2 as a new host cofactor that utilizes distinct regulatory domains to bind incoming HIV-1 particles and facilitate trafficking of incoming viral cores through MT stabilization. IMPORTANCE While microtubules (MTs) have long been known to be important for delivery of incoming HIV-1 cores to the nucleus, how the virus engages and exploits these filaments remains poorly understood. Our previous work revealed the importance of highly specialized MT regulators that belong to a family called plus-end tracking proteins (+TIPs) in facilitating early stages of infection. These +TIPs perform various functions, such as engaging cargos for transport or engaging peripheral actin to stabilize MTs, suggesting several family members have the potential to contribute to infection in different ways. Here, we reveal that cytoplasmic linker-associated protein 2 (CLASP2), a key regulator of cortical capture and stabilization of MTs, interacts with incoming HIV-1 particles, and we identify a distinct C-terminal domain in CLASP2 that promotes both MT stabilization and early infection. Our findings identify a new +TIP acting as a host cofactor that facilitates early stages of viral infection., (Copyright © 2020 American Society for Microbiology.)

HIV-1 uses the microtubule network to traffic the viral capsid core toward the nucleus. Viral nuclear trafficking and infectivity require the kinesin-1 adaptor protein FEZ1. Here, we demonstrate that FEZ1 directly interacts with the HIV-1 capsid and specifically binds capsid protein (CA) hexamers. FEZ1 contains multiple acidic, poly-glutamate stretches that interact with the positively charged central pore of CA hexamers. The FEZ1-capsid interaction directly competes with nucleotides and inositol hexaphosphate (IP6) that bind at the same location. In addition, all-atom molecular dynamic (MD) simulations establish the molecular details of FEZ1-capsid interactions. Functionally, mutation of the FEZ1 capsid-interacting residues significantly reduces trafficking of HIV-1 particles toward the nucleus and early infection. These findings support a model in which the central capsid hexamer pore is a general HIV-1 cofactor-binding hub and FEZ1 serves as a unique CA hexamer pattern sensor to recognize this site and promote capsid trafficking in the cell., (Published by Elsevier Inc.)

Being dependent upon host transport systems to navigate the cytoplasm, viruses have evolved various strategies to manipulate cytoskeletal functions. Generally, viruses use the actin cytoskeleton to control entry and short-range transport at the cell periphery and exploit microtubules (MTs) for longer-range cytosolic transport, in some cases to reach the nucleus. While earlier studies established the fundamental importance of these networks to successful infection, the mechanistic details and true extent to which viruses usurp highly specialized host cytoskeletal regulators and motor adaptors is only beginning to emerge. This review outlines our current understanding of how cytoskeletal regulation contributes specifically to the early stages of viral infection, with a primary focus on retroviruses and herpesviruses as examples of recent advances in this area., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Competing Interests: The author has declared that no competing interests exist.

Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The original version of this Article contained an error in the Methods section 'Viruses and drugs'. The timing for drug treatment of CHME3 4 × 4 or 293T cells with γ-secretase inhibitor or BACE1 inhibitor was incorrectly given as '1 day prior to infection or transfection' and should have stated '4 or 6 h post transfection or infection, respectively'. This error is now corrected in both the PDF and HTML versions of the Article.

While beta-amyloid (Aβ), a classic hallmark of Alzheimer's disease (AD) and dementia, has long been known to be elevated in the human immunodeficiency virus type 1 (HIV-1)-infected brain, why and how Aβ is produced, along with its contribution to HIV-associated neurocognitive disorder (HAND) remains ill-defined. Here, we reveal that the membrane-associated amyloid precursor protein (APP) is highly expressed in macrophages and microglia, and acts as an innate restriction against HIV-1. APP binds the HIV-1 Gag polyprotein, retains it in lipid rafts and blocks HIV-1 virion production and spread. To escape this restriction, Gag promotes secretase-dependent cleavage of APP, resulting in the overproduction of toxic Aβ isoforms. This Gag-mediated Aβ production results in increased degeneration of primary cortical neurons, and can be prevented by γ-secretase inhibitor treatment. Interfering with HIV-1's evasion of APP-mediated restriction also suppresses HIV-1 spread, offering a potential strategy to both treat infection and prevent HAND.

Although microtubule motors mediate intracellular virus transport, the underlying interactions and control mechanisms remain poorly defined. This is particularly true for HIV-1 cores, which undergo complex, interconnected processes of cytosolic transport, reverse transcription, and uncoating of the capsid shell. Although kinesins have been implicated in regulating these events, curiously, there are no direct kinesin-core interactions. We recently showed that the capsid-associated kinesin-1 adaptor protein, fasciculation and elongation protein zeta-1 (FEZ1), regulates HIV-1 trafficking. Here, we show that FEZ1 and kinesin-1 heavy, but not light, chains regulate not only HIV-1 transport but also uncoating. This required FEZ1 phosphorylation, which controls its interaction with kinesin-1. HIV-1 did not stimulate widespread FEZ1 phosphorylation but, instead, bound microtubule (MT) affinity-regulating kinase 2 (MARK2) to stimulate FEZ1 phosphorylation on viral cores. Our findings reveal that HIV-1 binds a regulatory kinase to locally control kinesin-1 adaptor function on viral cores, thereby regulating both particle motility and uncoating., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diaphanous (Dia)-related formins (DRFs) coordinate cytoskeletal remodeling by controlling actin nucleation and microtubule (MT) stabilization to facilitate processes such as cell polarization and migration; yet the full extent of their activities remains unknown. Here, we uncover two discrete roles and functions of DRFs during early human immunodeficiency virus type 1 (HIV-1) infection. Independent of their actin regulatory activities, Dia1 and Dia2 facilitated HIV-1-induced MT stabilization and the intracellular motility of virus particles. However, DRFs also bound in vitro assembled capsid-nucleocapsid complexes and promoted the disassembly of HIV-1 capsid (CA) shell. This process, also known as "uncoating," is among the most poorly understood stages in the viral lifecycle. Domain analysis and structure modeling revealed that regions of Dia2 that bound viral CA and mediated uncoating as well as early infection contained coiled-coil domains, and that these activities were genetically separable from effects on MT stabilization. Our findings reveal that HIV-1 exploits discrete functions of DRFs to coordinate critical steps in early infection and identifies Dia family members as regulators of the poorly understood process of HIV-1 uncoating., Competing Interests: The authors declare no conflict of interest.

Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (+TIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection., (Copyright © 2017 American Society for Microbiology.)

Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end-tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170-mediated capture to initiate transport in primary human cells., (© 2015 Jovasevic et al.)

Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. Although a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bi-directional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

Stable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs, and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant-negative inhibitor of MT stabilization prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although microtubules (MTs) frequently form highly dynamic networks, subsets of MTs become stabilized in response to environmental cues and function as specialized tracks for vesicle and macromolecular trafficking. MT stabilization is controlled by specialized plus-end tracking proteins (+TIPs) whose accumulation at the MT ends is facilitated by the end-binding protein, EB1, and regulated by various signaling pathways. As cargoes themselves, viruses are dependent on MTs for their intracellular movement. Although many viruses affect MT organization, the potential contribution of MT stabilization by +TIPs to infection remains unknown. Here we show that early in infection of primary human fibroblasts, herpes simplex virus type 1 (HSV-1) disrupts the centrosome, the primary MT organizing center in many cell types. As infection progresses HSV-1 induces the formation of stable MT subsets through inactivation of glycogen synthase kinase 3beta by the viral Ser/Thr kinase, Us3. Stable MT formation is reduced in cells infected with Us3 mutants and those stable MTs that form cluster around the trans-Golgi network. Downstream of glycogen synthase kinase 3beta, cytoplasmic linker-associated proteins (CLASPs), specialized host +TIPs that control MT formation at the trans-Golgi network and cortical capture, are specifically required for virus-induced MT stabilization and HSV-1 spread. Our findings demonstrate the biological importance of +TIPs to viral infection and suggest that HSV-1 has evolved to exploit the trans-Golgi network as an alternate MT organizing center to facilitate virus spread.

Many of the early events in retroviral infection are not well understood, but it is known that the host cytoskeleton and signaling pathways play integral roles in various entry and post-entry processes. Focal adhesion complexes act as sites of integration for both cytoskeletal organization and integrin signaling at the cell surface. Here, we show that talin-1 and vinculin, two interacting proteins that localize in focal adhesions to mediate integrin linkage to the actin cytoskeleton, function during retroviral infection. Transient overexpression of either talin-1 or vinculin reduced the susceptibility of human cells to infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus. In contrast, transient short interfering RNA-mediated knockdown of talin-1 or vinculin increased infection by pseudotyped HIV-1 and simian immunodeficiency virus, demonstrating that the endogenous forms of these proteins also impaired retroviral infection. Talin-1 or vinculin overexpression inhibited infection by retroviruses that entered the cell by either fusion or endocytosis, while analysis of HIV-1 DNA synthesis demonstrated that the block occurred early in infection and prior to the initiation of reverse transcription. Both factors retained antiviral activity in the presence of actin or microtubule depolymerizing agents. Finally, talin-1 and vinculin expression was found to negatively influence tyrosine phosphorylation of paxillin, a major focal adhesion scaffolding protein whose transient knockdown decreased pseudotyped HIV-1 infection. Together, these findings demonstrate that talin-1 and vinculin negatively affect tyrosine phosphorylation of paxillin, a novel positive regulator of HIV-1 infection, and impose an early block to infection by distinct retroviruses., (Published by Elsevier Ltd.)

The host cytoskeleton plays a central role in the life cycle of many viruses yet our knowledge of cytoskeletal regulators and their role in viral infection remains limited. Recently, moesin and ezrin, two members of the ERM (Ezrin/Radixin/Moesin) family of proteins that regulate actin and plasma membrane cross-linking and microtubule (MT) stability, have been shown to inhibit retroviral infection. To further understand how ERM proteins function and whether they also influence infection by other viruses, we identified PDZD8 as a novel moesin-interacting protein. PDZD8 is a poorly understood protein whose function is unknown. Exogenous expression of either moesin or PDZD8 reduced the levels of stable MTs, suggesting that these proteins functioned as part of a cytoskeletal regulatory complex. Additionally, exogenous expression or siRNA-mediated knockdown of either factor affected Herpes Simplex Virus type 1 (HSV-1) infection, identifying a cellular function for PDZD8 and novel antiviral properties for these two cytoskeletal regulatory proteins., (Published by Elsevier Inc.)

In a yeast two-hybrid screen for cellular factors that could interact with human immunodeficiency virus type 1 (HIV-1) Gag protein, we identified PDZD8 and confirmed the interaction by coimmunoprecipitation (co-IP). PDZD8 overexpression promoted the initiation of reverse transcription and increased infection by pseudotyped retroviruses independent of the route of viral entry, while transient knockdown of endogenous levels decreased HIV-1 infection. A mutant of PDZD8 lacking a predicted coiled-coil domain in its Gag-interacting region failed to bind Gag and promote HIV-1 infection, identifying the domain of PDZD8 required for mediating these effects. As such, we identify PDZD8 as a novel positive mediator of retroviral infection.

Neurons are one of the few cell types in the human body that do not support HIV type-1 (HIV-1) replication. Although the lack of key receptors is a major obstacle to infection, studies suggest that additional functions inhibit virus replication to explain the exquisite resistance of neurons to HIV-1. However, specific neuronal factors that may explain this resistance remain to be discovered. In a screen for antiviral factors using a fibroblast line chemically mutagenized and selected for resistance to retroviral infection, we recently identified induction of rat FEZ1 (fasciculation and elongation protein zeta-1), a brain-specific protein, as the cause of this resistance. When exogenously expressed in nonneuronal cell lines rat FEZ1 blocked nuclear entry of retroviral DNA. Here, we demonstrate that among human brain cells, neurons naturally express high levels of FEZ1 compared to astrocytes or microglia cells and are correspondingly less susceptible to infection with pseudotyped HIV-1 that bypasses receptor-mediated viral entry. Demonstrating that endogenous FEZ1 was functionally important in the resistance of neurons to HIV-1 infection, siRNA-mediated knockdown of endogenous FEZ1 increased the infectivity of neurons while sensitive brain cell types like microglia became more resistant upon FEZ1 overexpression. In addition, FEZ1 expression was not induced in response to IFN treatment. As such, in contrast to other widely expressed, IFN-inducible antiviral factors, FEZ1 appears to represent a unique neuron-specific determinant of cellular susceptibility to infection in a cell type that is naturally resistant to HIV-1.

We recently identified the cytoskeletal regulatory protein moesin as a novel gene that inhibits retroviral replication prior to reverse transcription by downregulation of stable microtubule formation. Here, we provide evidence that overexpression of ezrin, another closely related ezrin-radixin-moesin (ERM) family member, also blocks replication of both murine leukemia viruses and human immunodeficiency virus type 1 (HIV-1) in Rat2 fibroblasts before reverse transcription, while knockdown of endogenous ezrin increases the susceptibility of human cells to HIV-1 infection. Together, these results suggest that ERM proteins may be important determinants of retrovirus susceptibility through negative regulation of stable microtubule networks.

In the past decade, several lines of evidence have highlighted the importance of the host cell cytoskeleton in various stages of retroviral infection. To complete their lifecycle, retroviruses must penetrate the outer barrier of the cell membrane, and viral cores containing the viral genome must traverse the cytoplasm to the nucleus and then viral gene products must make the journey back to the cell surface in order to release new progeny. The presence of a dense cytoskeletal network and organelles in the cytoplasm creates an environment that greatly impedes diffusion of macromolecules such as viruses. As such, retroviruses have evolved means to hijack actin as well as microtubule cytoskeletal networks that regulate macromolecular movement within the host cell. Developing studies are discovering several host and viral factors that play important roles in retroviral trafficking.

In a functional screen of mammalian complementary DNA libraries, we identified moesin as a novel gene whose overexpression blocks infection by murine leukemia viruses and human immunodeficiency virus type 1 in human and rodent lines, before the initiation of reverse transcription. Knockdown of moesin by RNA interference resulted in enhanced infection, suggesting that even the endogenous basal levels of moesin in rat fibroblasts are sufficient to limit virus infection. Moesin acts as a crosslinker between plasma membrane and actin filaments, as well as a signal transducer in responses involving cytoskeletal remodeling. Moesin overexpression was found to downregulate the formation of stable microtubules, whereas knockdown of moesin increased stable microtubule formation. A virus-resistant mutant cell line also displayed decreased stable microtubule levels, and virus-sensitive revertants recovered from the mutant line showed restoration of the stable microtubules, suggesting that these cytoskeletal networks play an important role in early post-entry events in the retroviral lifecycle. Together, these results suggest that moesin negatively regulates stable microtubule networks and is a natural determinant of cellular sensitivity to retroviral infection.

Two mutant Rat2 fibroblast cell lines, R3-2 and R4-7, have been previously isolated by a selection for retrovirus resistance. We have now further analyzed the basis of the block to retroviral infection in the R3-2 line. Using Affymetrix GeneChip analysis, several genes were identified as differentially expressed in the mutant R3-2 line compared with the wild-type cells. One of the candidate gene products, FEZ1 (fasciculation and elongation protein zeta-1), a protein kinase C (PKC)zeta-interacting protein homologous to the Caenorhabditis elegans synaptic transport protein UNC-76, was found to be up-regulated >30-fold in the resistant R3-2 line. FEZ1 overexpression in Rat2 cells conferred a potent resistance to infection by genetically marked retroviruses, and the degree of retroviral resistance in both Rat2 fibroblasts and 293T cells tightly correlated with the expression level of FEZ1 transcripts. FEZ1-overexpressing Rat2 cells showed a similar phenotype to that of the mutant R3-2 line: Infection resulted in normal viral DNA synthesis but a reduction in the formation of circular DNA, indicating a block after reverse transcription but before nuclear entry. Partial knockdown of FEZ1 expression in R3-2 by RNA interference (RNAi) significantly reduced the resistance of this line to infection. Thus, our data suggest that FEZ1 overexpression is sufficient to explain the resistant phenotype of R3-2 cells and identify FEZ1 as a new gene capable of causing retrovirus resistance.

The enhancer region in the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) is very important for viral transcription. This promoter sequence binds both nuclear factor-kappaB and NFAT, two important modulators of HIV-1 gene expression. Previous studies have indicated that the enhancer regions of the different HIV-1 clade LTRs differ in their number of NF-kappaB-binding sites. In this study, we have compared the activation potential of the different HIV-1 clade and HIV-2 LTRs and assessed their interaction with NFAT and NF-kappaB. In T-cell lines and primary CD4(+) T-cells, the results showed that the HIV-1 clade E LTR (with a single NF-kappaB-binding site) was the weakest LTR regardless of the tested activators, whereas the HIV-2 LTR was the most responsive LTR. The clade E enhancer region was also demonstrated to be the weakest enhancer region in transfection experiments with luciferase reporter-based vectors. Electrophoretic mobility shift assays with extracts from activated CD4(+) T-cells indicated that, although NF-kappaB and NFAT bound all enhancers, HIV-1 clade E and HIV-2 LTR enhancers were poor binding targets for these two factors. Weak NFAT binding to clade E enhancers was also confirmed using NFAT1-expressing 293T cells in competition experiments. We have also shown the absence of interaction of NF-kappaB or NFAT with the third NF-kappaB repeat present in clade C. However, the clade C enhancer bound NFAT more efficiently than all other enhancer regions tested. Our results hence demonstrate for the first time that differences in the binding of NF-kappaB and NFAT to the enhancer regions could be responsible for some of the observed variation in HIV-1 clade LTR activation, whereas HIV-2 LTR activation seems mostly independent of these interactions.

We investigated whether the high mobility group B 1 (HMGB1), an abundant nuclear protein in all mammalian cells, affects HIV-1 transcription. Intracellular expression of human HMGB1 repressed HIV-1 gene expression in epithelial cells. This inhibitory effect of HMGB1 was caused by repression of long terminal repeat (LTR)-mediated transcription. Other viral promoters/enhancers, including simian virus 40 or cytomegalovirus, were not inhibited by HMGB1. In addition, HMGB1 inhibition of HIV-1 subtype C expression was dependent on the number of NF kappa B sites in the LTR region. The inhibitory effect of HMGB1 on viral gene expression observed in HeLa cells was confirmed by an upregulation of viral replication in the presence of antisense HMGB1 in monocytic cells. In contrast to what was found in HeLa cells and monocytic cells, endogenous HMGB1 expression did not affect HIV-1 replication in unstimulated Jurkat cells. Thus, intracellular HMGB1 affects HIV-1 LTR-directed transcription in a promoter- and cell-specific manner.

Positive transcription elongation factor-b (P-TEFb) contains CDK9 and cyclin T(1). P-TEFb was affinity purified from a stably transfected cell line that expresses epitope-tagged CDK9, and proteins that appeared to be specifically bound were sequenced. In addition to CDK9, previously identified isoforms of cyclin T (including T(1), T(2A) and T(2B)), HSP90 and CDC37, this analysis identified a novel protein named MCEF. Cloning of its cognate cDNA revealed that MCEF is the newest member of the AF4 family of transcription factors involved in acute lymphoblastic leukemia. MCEF RNA was expressed in all human tissues examined, and antisera directed against recombinant MCEF specifically immunoprecipitated P-TEFb. Ectopic expression of MCEF did not activate HIV-1 replication, and tethering of MCEF to a promoter did not activate transcription., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)

Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated. In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF. In vitro binding assays showed that recombinant USF(43) interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.

Objective: To determine whether short peptides corresponding to the RGPGR motif of the V3 loop of gp 120 have anti-human immunodeficiency virus type 1 (anti-HIV-1) activity., Design/methods: Short peptides were tested against the HIV-1 laboratory strains and clinical isolates., Results: The tripeptide glycyl-prolyl-glycine amide (GPG-NH2) inhibited the replication of both laboratory strains and 47 clinical isolates, including 19 strains that were resistant to other drugs or that were from patients with failing therapy. The 50% inhibitory concentrations values were 2.7 to 37 microM. Phenotypic change of two isolates from nonsyncytia-inducing to syncytia-inducing did not change their sensitivity to GPG-NH2. The tripeptide added to the antiviral effect of both zidovudine and ritonavir., Conclusions: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action. Glycyl-prolyl-glycine-NH2 might prove useful by itself or as a lead compound for the treatment of drug-resistant HIV-1. Glycyl-prolyl-glycine-NH2 is currently undergoing phase I/II human clinical trials in Sweden.

Objective: To determine whether the peptide glycyl-prolyl-glycine amide (GPG-NH2) corresponding to a conserved motif in the tip of the third hypervariable region of gp120 affected the early events in the human immunodeficiency virus type 1 (HIV-1) replication., Design/methods: Glycyl-prolyl-glycine amide was tested for its effect on HIV-1 adsorption, co-receptor usage, proviral DNA synthesis, messenger RNA (mRNA) synthesis and splicing, translation, tat/TAR transactivation, and virus protease activity., Results: Glycyl-prolyl-glycine amide did not appear to affect the early events of the virus replication. HIV-1 having glycine-leucine-glycine instead of GPG in the V3 loop and the mutants deleted of the GPG motif were still inhibited by the peptide. Glycyl-prolyl-glycine-NH2 had no discernible effect on any of the other steps in the virus replication cycle tested. The only effect observed was an increased sodium dodecyl sulfate polyacrylamide amide gel electrophoresis mobility of gp160/120 at high concentrations of GPG-NH2., Conclusions: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action.

Transcription of the HIV-1 provirus genome is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral long terminal repeat (LTR) region of HIV-1. However, several cellular transcription factors have been identified that can interact with the HIV-1 LTR; the significance of all of these factors is not clearly understood. In this study we have characterized the LTR region of different subtypes of HIV-1 with regard to nucleotide sequence and promoter activity. The LTR regions of HIV-1 from peripheral blood mononuclear cells of 29 infected individuals originating from 10 different geographical regions were sequenced and further analyzed for promoter/enhancer activity in transient transfection of HeLa cells, in the context of a reporter gene and in the context of the complete virus genome. We found several subtype-specific LTR sequences of the various HIV-1 strains, such as an insertion that created a potential third NF-kappaB site in the LTR of the subtype C strains. The USF-binding site in the NRE also contained subtype-specific sequences. Interestingly, the promoter/enhancer activities of the subtype C LTRs were higher than the activities of the other subtypes analyzed here (subtypes A, B, D, E, and G), suggesting that the potential third NF-kappaB site may confer higher LTR activity or that the subtype C NRE may be less potent. Thus, our data suggest that genetic diversity of the LTR may result in HIV-1 subtypes with different replicative properties.