Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system.

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission.

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint.

Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission.

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that is used to recruit essential cofactors to sites of ESCRT activity.

Recurrent evolution of an inhibitor of ESCRT-dependent virus budding and LINE-1 retrotransposition in primates.

Most antiviral proteins recognize specific features of viruses. In contrast, the recently described antiviral factor retroCHMP3 interferes with the "host endosomal complexes required for transport" (ESCRT) pathway to inhibit the budding of enveloped viruses. RetroCHMP3 arose independently on multiple occasions via duplication and truncation of the gene encoding the ESCRT-III factor CHMP3.

Stephan Oroszlan and the Proteolytic Processing of Retroviral Proteins: Following A Pro.

Steve Oroszlan determined the sequences at the ends of virion proteins for a number of different retroviruses. This work led to the insight that the amino-terminal amino acid of the mature viral CA protein is always proline. In this remembrance, we review Steve's work that led to this insight and show how that insight was a necessary precursor to the work we have done in the subsequent years exploring the cleavage rate determinants of viral protease processing sites and the multiple roles the amino-terminal proline of CA plays after protease cleavage liberates it from its position in a protease processing site.

RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice.

Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing.

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop during an active checkpoint.

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release.

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined.

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress.

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined.

Membrane Remodeling: ESCRT-III Filaments as Molecular Garrotes.

New reconstitution, biochemical and structural studies are revealing how the core machinery of the ESCRT pathway constricts membranes to promote fission. Equally exciting is the discovery and characterization of conserved ESCRT-like machinery across all three domains of life.

Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro.

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells.

Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections.

Membrane constriction and thinning by sequential ESCRT-III polymerization.

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood.

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use.

Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases.

Many AAA+ ATPases form hexamers that unfold protein substrates by translocating them through their central pore. Multiple structures have shown how a helical assembly of subunits binds a single strand of substrate, and indicate that translocation results from the ATP-driven movement of subunits from one end of the helical assembly to the other end. To understand how more complex substrates are bound and translocated, we demonstrated that linear and cyclic versions of peptides bind to the AAA+ ATPase Vps4 with similar affinities, and determined cryo-EM structures of cyclic peptide complexes.

Retrovirus Restriction by TRIM5α: RINGside at a Cage Fight.

The restriction factor TRIM5α recognizes incoming retroviral capsids and blocks virus replication. In this issue of Cell Host & Microbe, Fletcher et al. (2018) show how the TRIM5α RING domain mediates formation of various ubiquitin conjugates that differentially regulate TRIM5α turnover, inhibition of viral reverse transcription, and innate immune activation.

A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability.

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4C) increases cancer susceptibility.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes.

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II.

General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity ( of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition.

The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets.

The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe et al.

What's in a name?

Numerous concerns have been raised about the sustainability of the biomedical research enterprise in the United States. Improving the postdoctoral training experience is seen as a priority in addressing these concerns, but even identifying who the postdocs are is made difficult by the multitude of different job titles they can carry. Here, we summarize the detrimental effects that current employment structures have on training, compensation and benefits for postdocs, and argue that academic research institutions should standardize the categorization and treatment of postdocs.

Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase.

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeF, and an ESCRT-III substrate peptide.

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells.

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In , deletion of the ATPase leads to severe defects in nuclear morphology and integrity.

Designed proteins induce the formation of nanocage-containing extracellular vesicles.

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses.