Cryo-EM Structure of Dodecameric Vps4p and Its 2:1 Complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly

Three-dimensional Structure of HIV-1 Virus-like Particles by Electron Cryotomography

While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about how the individual proteins are arranged in the mature infectious viral particle. Here, we report the three-dimensional structures of individual HIV-1 virus-like particles (VLPs) as obtained by electron cryotomography. These reconstructions revealed that while the structures and positions of the conical cores within each VLP were unique, they exhibited several surprisingly consistent features, including similarities in the size and shape of the wide end of the capsid (the “base”), uniform positioning of the base and other regions of the capsid 11 nm away from the envelope/MA layer, a cone angle that typically varied from 24° to 18° around the long axis of the cone, and an internal density (presumably part of the NC/RNA complex) cupped within the base. Multiple and nested capsids were observed. These results support the fullerene cone model for the viral capsid, indicate that viral maturation involves a free re-organization of the capsid shell rather than a continuous condensation, imply that capsid assembly is both concentration-driven and template-driven, suggest that specific interactions exist between the capsid and the adjacent envelope/MA and NC/RNA layers, and show that a particular capsid shape is favored strongly in-vivo

Toward a sustainable biomedical research enterprise: Finding consensus and implementing recommendations

The US research enterprise is under significant strain due to stagnant funding, an expanding workforce, and complex regulations that increase costs and slow the pace of research. In response, a number of groups have analyzed the problems and offered recommendations for resolving these issues. However, many of these recommendations lacked follow-up implementation, allowing the damage of stagnant funding and outdated policies to persist. Here, we analyze nine reports published since the beginning of 2012 and consolidate over 250 suggestions into eight consensus recommendations made by the majority of the reports. We then propose how to implement these consensus recommendations, and we identify critical issues, such as improving workforce diversity and stakeholder interactions, on which the community has yet to achieve consensus

Point of View: 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. We also present brief case studies of two institutions that have addressed these challenges and can provide models for other institutions attempting to enhance their postdoctoral workforces and improve the sustainability of the biomedical research enterprise

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 Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the “late domain”) binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs; residues 222–777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222–777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding

Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal Domain of the Viral CA Protein

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action

General model for retroviral capsid pattern recognition by TRIM5 proteins

Permission to archive final published versionRestriction 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 (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognitionYe

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