Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

Cells respond to stress through adaptive mechanisms that limit cellular damage and prevent cell death. MicroRNAs act as regulators of stress responses and stress can impact the functioning of miRNA biogenesis pathways. We were interested in the effect that severe proteotoxic stress capable of inducing apoptosis may have on miRNA biogenesis and the impact of the molecular chaperone protein HSP70 under these conditions. We found that the miRNA processing enzymes Drosha and Dicer and their accessory proteins DGCR8 and TRBP2 are cleaved by caspases in apoptotic cells. Overexpression of HSP70 prevented caspase activation and the degradation of these processing proteins. Caspase cleavage of TRBP2 was mapped to amino acid 234 which separates the two dsRNA-binding domains from the C-terminal Dicer interacting domain. Overexpression of TRBP2 was found to increase miRNA maturation, while expression of either of the fragments generated by caspase cleavage impaired maturation. These results indicate that inactivation of miRNA biogenesis is a critical feature of apoptosis and that cleavage of TRBP2, rather than simply a loss of function, serves to create positive acting inhibitors of pre-miRNA maturation.

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 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.