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
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.
Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to phosphoinositide lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and bound tubulin dimers (kD = 1.89 µM) to impact microtubule dynamics. In PFN1-deficient mammalian cells, Halo-PFN1 or mApple-PFN1 (mAp-PEN1) restored morphological and cytoskeletal functions. Titrations of self-labeling Halo-ligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.