Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology.

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.

Using antiubiquitin antibodies to probe the ubiquitination state within rhTRIM5α cytoplasmic bodies.

The first line of defense protecting rhesus macaques from HIV-1 is the restriction factor rhTRIM5α, which recognizes the capsid core of the virus early after entry and normally blocks infection prior to reverse transcription. Cytoplasmic bodies containing rhTRIM5α have been implicated in the ubiquitin-proteasome pathway, but the specific roles these structures play remain uncharacterized. Here, we examine the ubiquitination status of cytoplasmic body proteins. Using antibodies specific for different forms of ubiquitin, we show that ubiquitinated proteins are present in cytoplasmic bodies, and that this localization is altered after proteasome inhibition. A decrease in polyubiquitinated proteins localizing to cytoplasmic bodies was apparent after 1 h of proteasome inhibition, and greater differences were seen after extended proteasome inhibition. The decrease in polyubiquitin conjugates within cytoplasmic bodies was also observed when deubiquitinating enzymes were inhibited, suggesting that the removal of ubiquitin moieties from polyubiquitinated cytoplasmic body proteins after extended proteasome inhibition is not responsible for this phenomenon. Superresolution structured illumination microscopy revealed finer details of rhTRIM5α cytoplasmic bodies and the polyubiquitin conjugates that localize to these structures. Finally, linkage-specific polyubiquitin antibodies revealed that K48-linked ubiquitin chains localize to rhTRIM5α cytoplasmic bodies, implicating these structures in proteasomal degradation. Differential staining of cytoplasmic bodies seen with different polyubiquitin antibodies suggests that structural changes occur during proteasome inhibition that alter epitope availability. Taken together, it is likely that rhTRIM5α cytoplasmic bodies are involved in recruiting components of the ubiquitin-proteasome system to coordinate proteasomal destruction of a viral or cellular protein(s) during restriction of HIV-1.

Unclosed HIV-1 capsids suggest a curled sheet model of assembly.

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms.

Recruitment and dynamics of proteasome association with rhTRIM5α cytoplasmic complexes during HIV-1 infection.

A variety of proteins have been identified that restrict infection by different viruses. One such restriction factor is the rhesus macaque variant of TRIM5α (rhTRIM5α), which potently blocks infection by HIV-1. The block to infection mediated by rhTRIM5α occurs early after entry into the host cell, generally prior to reverse transcription. However, proteasome inhibitors reveal an intermediate step of restriction in which virus can complete reverse transcription, but still fails to infect the cell. While proteasome inhibitors have been a useful tool in understanding how restriction takes place, the role of the proteasome itself during restriction has not yet been examined. Here, we characterize the interaction of rhTRIM5α and incoming virions with the proteasome. We show that proteasomes localize to rhTRIM5α cytoplasmic bodies, and this localization is more evident when the activity of the proteasome is inhibited pharmacologically. We also show that restricted virus associates with complexes of proteasomes and rhTRIM5α, suggesting that rhTRIM5α utilizes the proteasome during restriction. Finally, live cell imaging experiments reveal that virus associates with proteasomes, and proteasome inhibition affects the duration of association. Taken together, these studies implicate the proteasome as playing a functional role during rhTRIM5α restriction of incoming virions.

Identification of residues within the L2 region of rhesus TRIM5alpha that are required for retroviral restriction and cytoplasmic body localization.

The intracellular restriction factor TRIM5alpha, inhibits infection by numerous retroviruses in a species-specific manner. The best characterized example of this restriction is the TRIM5alpha protein from rhesus macaques (rhTRIM5alpha), which potently inhibits HIV-1 infection. TRIM5alpha localizes to cytoplasmic assemblies of protein referred to as cytoplasmic bodies, though the role that these bodies play in retroviral restriction is unclear. We employed a series of truncation mutants to identify a discrete region, located within the Linker2 region connecting the coiled-coil and B30.2/PRYSPRY domains of TRIM5alpha, which is required for cytoplasmic body localization. Deletion of this region in the context of full-length rhTRIM5alpha abrogates cytoplasmic body localization. Alanine mutagenesis of the residues in this region identifies two stretches of amino acids that are required for both cytoplasmic body localization and retroviral restriction. This work suggests that the determinants that mediate TRIM5alpha localization to cytoplasmic bodies play a requisite role in retroviral restriction.

Imaging of HIV/host protein interactions.

HIV-1 relies on a myriad of interactions with host cell proteins to carry out its life cycle. Traditional biochemical approaches to probe protein-protein interactions are limited in their ability to study the spatial and dynamic interactions that take place in the context of an intact cell. However, issues such as localization and dynamics of interactions between viral and host proteins can be well addressed utilizing fluorescent imaging methods. The past decade has brought about the development of many novel fluorescent imaging techniques which have proved useful to describe the interaction of HIV-1 proteins with the host cell.