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.

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.

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.

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.

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.

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.