Abstract
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, 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.
Highlights
Productive infection by the human immunodeficiency virus 1 (HIV-1), the causative agent of AIDS, requires reverse transcription (RT) of the viral RNA genome into double-stranded viral DNA and subsequent integration of the vDNA into host cell chromosomes
Studies in immortalized cell lines such as HeLa and activated CD4+ T cells have established a spatiotemporal sequence of events where: (i) vDNA is synthesized by RT in the cytoplasm, with concomitant degradation of the template viral RNA (vRNA), (ii) the vDNA genome translocates into the nucleus, (iii) the integrase enzyme (IN) inserts the vDNA into the genome, and (iv) the integrated vDNA genome undergoes transcription that leads to viral progeny (Freed, 2001; Hu & Hughes, 2012)
We demonstrated that vDNA and vRNA genomes cluster together in nuclear niches associated with speckle factors and that these clusters can form in absence of viral integration into the host genome
Summary
Productive infection by the human immunodeficiency virus 1 (HIV-1), the causative agent of AIDS, requires reverse transcription (RT) of the viral RNA (vRNA) genome into double-stranded viral DNA (vDNA) and subsequent integration of the vDNA into host cell chromosomes. Macrophages are terminally differentiated, non-dividing cells derived from blood monocytes, which play a critical role in the innate and adaptive immune response (Gordon & Taylor, 2005; Koppensteiner et al, 2012; Ganor et al, 2019). Along with activated CD4+ T cells, macrophages are natural target cells for HIV-1, and accumulating evidence points to a critical role of these cells in viral persistence, which remains a major roadblock to eradicating HIV (Honeycutt et al, 2017; Ganor et al, 2019). Despite this importance, the early steps of HIV-1 infection in macrophages remain elusive. Our data reveal that genomic vRNA forms nuclear clusters that associate with nuclear speckle factors and provide surprising evidence that these structures can harbor a nuclear RT activity
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