HIV-1 promotes ubiquitination of the amyloidogenic C-terminal fragment of APP to support viral replication.

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.

Microtubules and viral infection.

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.

FEZ1 Plays Dual Roles in Early HIV-1 Infection by Independently Regulating Capsid Transport and Host Interferon-Stimulated Gene Expression.

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-ß treatment on early stages of HIV-1 infection in different cell systems with various IFN-ß 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-ß 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-ß'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.

Virus update for the M2 "mac-in-touch".

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.

The host cytoskeleton: a key regulator of early HIV-1 infection.

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.

FEZ1 phosphorylation regulates HSPA8 localization and interferon-stimulated gene expression.

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.

Dynactin 1 negatively regulates HIV-1 infection by sequestering the host cofactor CLIP170.

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.

HIV-1 capsid exploitation of the host microtubule cytoskeleton during early infection.

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.

HIV-1 capsids mimic a microtubule regulator to coordinate 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.

HIV-1 Exploits CLASP2 To Induce Microtubule Stabilization and Facilitate Virus Trafficking to the Nucleus.

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.

FEZ1 Is Recruited to a Conserved Cofactor Site on Capsid to Promote HIV-1 Trafficking.

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.

Exploitation of Cytoskeletal Networks during Early Viral Infection.

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.

Poxviruses Evade Cytosolic Sensing through Disruption of an mTORC1-mTORC2 Regulatory Circuit.

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.

Author Correction: HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration.

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.

HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration.

While beta-amyloid (Aß), 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 Aß 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 Aß isoforms. This Gag-mediated Aß 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.

Localized Phosphorylation of a Kinesin-1 Adaptor by a Capsid-Associated Kinase Regulates HIV-1 Motility and Uncoating.

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.

Distinct functions of diaphanous-related formins regulate HIV-1 uncoating and transport.

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.

Microtubule Regulation and Function during Virus Infection.

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.

Microtubule plus end-associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells.

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.