Extremophiles, microorganisms thriving in extreme environmental conditions must have proteins and nucleic acids that are stable at extremes of temperature and pH. The non-enveloped, rod-shaped virus SIRV2 infects the hyperthermophilic acidophile Sulfolobus islandicus which lives at 80° C and pH 3. We have used cryo-EM to generate a three-dimensional reconstruction of the SIRV2 virion at ~ 4 Å resolution, which revealed a novel form of virion organization. While almost half of the capsid protein is unstructured in solution, this unstructured region folds in the virion into a single extended α-helix which wraps around the DNA. The DNA is entirely in A-form, suggesting a common mechanism with bacterial spores for protecting DNA in the most adverse environments.

Single molecule localization microscopy can generate 3D super-resolution images without scanning by leveraging the axial variations of normal or engineered point spread functions (PSF). Successful implementation of these approaches for extended axial ranges remains, however, challenging. We present Zernike Optimized Localization Approach in 3D (ZOLA-3D), an easy-to-use computational and optical solution that achieves optimal resolution over a tunable axial range. We use ZOLA-3D to demonstrate 3D super-resolution imaging of mitochondria, nuclear pores and microtubules in entire nuclei or cells up to ~5 μm deep.

In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA (vRNA), in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.