Cells have many ways to fight pathogens, but they still manage to evade our defenses. HIV, for example, is able to enter a cell, cross the cytoplasm, and then pass through the nuclear pore complex to reach the cell nucleus. The DNA within the virus is surrounded by a capsid that protects against defense mechanisms of the host, such as nucleic acid sensors and nucleases (Yan et al., 2010; Jacques et al., 2016; Francis and Melikyan, 2018; Burdick et al., 2017; Rasaiyaah et al., 2013).
Previously, it was thought that the capsid cannot enter the nucleus, but demonstration that the capsid depends on a nuclear protein called CPSF6 suggested that it might (Price et al., 2014; Lee et al., 2010). Now, in eLife, Hans-Georg Kräusslich and colleagues – including David Alejandro Bejarano and Ke Peng as joint first authors – report new insights into the role of CPSF6 and the entry of the capsid into the nucleus (Bejarano et al., 2019). The researchers – who are based at the University of Heidelberg and the Chinese Academy of Sciences – tagged HIV molecules with fluorescent markers and observed how HIV infected macrophages. The capsids and CPSF6 proteins were labeled with antibodies.
Bejarano et al. found that, contrary to previous assumptions, CPSF6 is essential for HIV to travel through the nuclear pore into the nucleus of macrophages. They also found that CPSF6 associated with capsids inside the nucleus, suggesting that intact capsids may cross the nuclear pore. When CPSF6 was depleted, or could no longer bind to the capsid, the virus accumulated outside the nucleus. This indicates a key role for CPSF6 in viral nuclear entry. However, similar to previous experiments, removing CPSF6 only modestly reduced the infection levels in macrophages because the capsids used the remaining low CPSF6 levels.
It is known that the capsid also binds a nuclear pore protein called Nup153 (Price et al., 2014). Bejarano et al. suggest that the capsid first interacts with Nup153 inside the nuclear pore complex; Nup153 is then displaced by nuclear CPSF6, which helps to pull the capsid inside the nucleus (Figure 1). They also propose an intriguing model to explain how the viral DNA could integrate into host DNA in the absence of CPSF6. In line with the hypotheses proposed in other studies (Achuthan et al., 2018; Schaller et al., 2011; Sowd et al., 2016), Bejarano et al. suggest that, in the absence of CPSF6, the capsid pokes through the nuclear pore complex enough for the integration of DNA to take place at the periphery of the nucleus.
Two key questions remain. First, why is CPSF6 required for replication in macrophages but not in cell lines? One reason for this might be that, in the absence of binding to a cofactor such as CPSF6, HIV-1 triggers the production of interferon in macrophages (Rasaiyaah et al., 2013). We can think of this in terms of cell lines 'licensing' otherwise 'illegal' behaviors by viruses. For example, capsid uncoating in the cytoplasm is 'illegal' in macrophages: that is, it does not lead to infection. However, such behaviour is licensed in cell lines and results in infection. This might be because, in macrophages, interferon suppresses mutant HIV-1 infection and uncoated cytosolic capsids are degraded. In cell lines, on the other hand, these 'illegal' viral behaviours are allowed and the virus can replicate.
Second, what is the role of CPSF6 in T cells (the immune cells that are the main target of HIV)? Given their small size and minimal cytoplasm, T cells are less suitable for microscopy, although nuclear transport can be studied in them (Achuthan et al., 2018). A better understanding of how HIV replicates in T cells, and where capsid uncoating occurs, may reveal fundamental differences in infection mechanisms between T cells and macrophages. In turn, this may help us understand the relative importance of HIV replication in macrophages and T cells in vivo.
This elegant study is a real technical tour de force, which has overcome significant experimental challenges to answer key questions in the field. As the techniques used by Bejarano et al. become widely adopted, we look forward to rapid advances in our understanding of the basic biology of HIV that may ultimately lead to more effective ways to treat, and eventually cure, HIV and AIDS.
- Version of Record published: March 5, 2019 (version 1)
© 2019, Zuliani-Alvarez and Towers
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Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca2+ concentrations oscillate in two approaching hyphae, and depletion of Ca2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion.
The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an ‘Escaping’ effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for ‘Escaping’. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.