Trafficking Avenir Group
- Toward the nucleus -
Projects
The main interest of our research group is focused on the early steps of Human Immunodeficiency Virus type 1 replication, from entry into the target cell to integration in the host chromatin.
HIV-1 uncoating
Although most agree that HIV uncoating occurs after fusion-dependent entry in the cytoplasm and before nuclear import, the field remains divided as to the precise moment and location for this event. We have previously shown that uncoating occurs upon completion of reverse transcription (Arhel et al., EMBO J 2007). Since transport to the nuclear envelope is very rapid (several minutes) compared to the reverse transcription process (several hours), this implies that uncoating occurs after docking at the nuclear pore. Indeed, using in situ scanning electron microscopy, we observed intact capsid cores docked at the nuclear pores at early time points post-infection (Arhel et al., EMBO J. 2007). More recently, we used PALM super-resolution imaging to visualise the distribution of integrase within capsid cores and noted that integrase clusters were larger in the cytoplasm than in the nucleus, and of a similar size to capsid cores (Lelek et al., PNAS 2012), thus confirming that cytoplasmic integrase is largely present in intact capsids and that these can be found deep within the cytoplasm. Uncoating is induced prematurely by restriction factors of the TRIM5alpha family of proteins. Rhesus macaques are resistant to infection by HIV-1 as a result of an innate cellular restriction mechanism attributable to the expression of rhTRIM5alpha, a member of the large tripartite motif (TRIM) protein family.
Viral trafficking

HIV does not have intrinsic motility and diffusion through the cytoplasm is inefficient and rarely leads to the required destination. Therefore, HIV can only move inside its target cell by usurping the existing cellular machinery.
Trafficking of incoming retroviruses along microtubules is likely to involve molecular motors of both the dynein and kinesin families since single particle tracking in living cells exposed to fluorescent viruses revealed bi-directional microtubular displacement, characterized by a series of saltatory retrograde and anterograde movements with overall directionality towards the nuclear compartment. Retroviruses could therefore bind to molecular motors of opposite polarity sequentially or simultaneously as has been proposed for HSV-132, and undergo a tug-of-war phenomena with overall movement towards the center of the cell. The (-)-ended motor dynein is thought to account for retroviral movement towards the nucleus since treatment with the dominant negative inhibitor p150 CC1 domain of dynactin (which uncouples dynein-based transport) or microinjection of anti-dynein antibodies, lead to a clustering of viruses in the cell periphery. A direct interaction between HFV Gag and LC8, light chain of dynein, has been demonstrated. However, it is not certain how relevant this interaction is for dynein-based transport and no such interaction has been demonstrated for HIV. Similarly, although microfilaments are also thought to be involved in trafficking of incoming HIV, no functional interaction with myosin motors has been demonstrated. This could be due on the one hand to the size and complexity of motors, which comprise several heavy and light chains and work in conjunction with other proteins, to the functional redundancy of family members (particularly for kinesins), or on the other hand to the fragile nature of the HIV capsid, which renders interaction approaches complex.
HIV-1 nuclear import
The unique ability of HIV-1 and other lentiviruses to efficiently replicate in non-dividing cells results from their ability to use of an active nuclear import strategy allowing the DNA genome to cross the nuclear pore of an intact nuclear membrane to gain access to and integrate into the cellular chromatin. The mitosis-independent replication of lentiviruses is a key feature to the development of lentiviral-derived gene transfer vectors with promising therapeutic applications.
We initially focused our studies on the viral determinants of HIV-1 active nuclear import, and particularly on the role of the HIV-1 central DNA Flap, which is a cis-acting sequence that is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The DNA Flap is a determinant of nuclear import but experiments carried out with cPPT/CTS double mutant viruses also point to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication (Iglesias et al., Retrovirology 2011).
More recently, we have turned our focus on the cellular proteins and pathways that the virus uses to efficiently cross the nuclear pore. Although several viral elements have been proposed to act as determinants of HIV-1 nuclear import (e.g. integrase, DNA Flap), evidence suggests that HIV-1 depends on host cell proteins to achieve translocation. Previous studies have shown the implication of several nucleoporins (Nup62, Nup85, Nup98, Nup107, Nup133, Nup153, Nup160, Nup214/CAN, and Nup358/RanBP2) in HIV-1 nuclear import and/or infectivity. However, the mechanistic implication and individual contribution of these apparently redundant functions remain to be clarified. In particular, the physiological implication of many nucleoporins in gene transcription and export of RNAs from the nucleus means that their knock-down can have important effects on read-outs of infectivity without actually participating in viral translocation through the nuclear pore. We have shown that Nup358/RanBP2, which is the main constituent of nuclear pore cytoplasmic filaments, serves as docking point for incoming HIV-1 capsids at the nuclear pore and that this interaction is in part mediated by a cyclophilin-homology domain present at the C-terminus of the protein (Di Nunzio et al., Plos One 2012). We are currently focusing our efforts on better understanding how HIV-1 achieves actual translocation across the nuclear pore lumen.