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Abstract

Upon fusion of the viral envelope with the host cell membrane, the capsid of the human immunodeficiency virus 1 (HIV-1) is released into the host cell cytoplasm. To productively infect a cell, the viral RNA genome needs to be reverse transcribed into viral DNA. This in turn needs to become integrated into the host cell genome. Integration can, however, only happen, after the viral genome is released from its capsid-container, in a process called uncoating.

This is a vital process and needs to be regulated and orchestrated in certain ways – which are still elusive and controversially discussed.

Some studies suggest that uncoating takes place soon after -, or concomitant with viral entry. Other researchers came to the result that the capsid needs to retain its structure to shield the viral components from being sensed by the innate cellular immune system. Both hypotheses, early uncoating and prolonged structural retention, are solidly supported by experimental data. Therefore, the timing and kinetics of uncoating remain unresolved. Based on previous results from our group, we had reason to believe that the capsid might indeed be retained, possibly even within the nucleus. A method was developed, that allows the detection of viral DNA. The presence of viral DNA was used as a criterion to discriminate between productive and nonproductive subviral particles in infected cells. Surprisingly, productive subviral particles displayed an intense, stable signal for capsid protein in immunofluorescence experiments, throughout the cytoplasm and even within the nuclei of infected cells. A strong signal is can be understood as a high concentration of labeled protein, which in turn might indicate the presence of a retained structure. However, intense immunofluorescence signals can also mean more efficient binding of antibodies due to structural rearrangements (such as uncoating), and a high spatial concentration of proteins cannot be directly interpreted as structure retention.

In this study, we present a unique way to address and solve this important question. We specifically focused on the small fraction of productive particles. Light Microscopy allows specific labeling but has low resolution. Electron Microscopy yields much higher resolution, but specific (immuno)labeling is difficult and often detrimental to ultrastructural retention. We overcame both limitations by correlative light – and electron microscopy: Regions of interest were identified by specific nuclear subviral particle surrogate markers in light microscopy. On these regions, tilt series electron tomography was performed, to visualize the subviral particles’ structure, as well as the subcellular environment, around the region of interest.

Performing high resolution tilt series electron tomography, we could repeatedly and convincingly visualize a capsid-reminiscent structure that underlies HIV-1 nuclear preintegration complexes. This apparent structure is very similar in shape, but smaller in size compared to capsids of virus particles of mostly identical preparations.

The discovery of a retained capsid structure in the nucleus of an infected cell will advance on our understanding of nuclear entry and provides whole new insights into the overall understanding of HIV-1 in early steps of infection.