The autophagy-related protein ATG5 is a central mediator of a non-canonical autophagy pathway hijacked by HIV-1 to weaken the host’s response to infection

ABSTRACT

Understanding how viruses evade innate defenses to efficiently spread in their hosts is crucial in the fight against infections. In our study, we provided new insights on the first step initiating an LC3C (microtubule associated protein 1 light chain 3 gamma)-associated degradative pathway exploited by HIV-1 (human immunodeficiency virus type 1) to overcome the antiviral action of the restriction factor BST2 (bone marrow stromal cell antigen 2)/tetherin. We have uncovered an unsuspected and unconventional function of the autophagy-related protein ATG5 in the recognition and engagement of BST2 molecules trapping viruses at the plasma membrane, and directing them toward this LC3C-associated pathway for degradation. Additionally, we highlighted that HIV-1 uses this LC3C-associated process to attenuate the inflammatory responses triggered by BST2-mediated sensing of viruses.

HIV-1 (Human immunodeficiency virus type 1), the virus causing AIDS (acquired immunodeficiency syndrome) remains a major global public health issue, with over 650,000 deaths attributed to its infection in 2021. In the battle against the spread of HIV-1 but also other viruses, the host immune system has developed an arsenal of defenses, including intrinsic cellular mechanisms that involve complex interactions between viral and cellular proteins. Host restriction factors are an important facet of these innate defenses against the virus. Amongst them, BST2 (bone marrow stromal cell antigen 2)/tetherin exerts its antiviral activity on numerous enveloped viruses by physically retaining newly formed viral particles at the surface of infected cells, efficiently blocking their release and subsequent dissemination. Additionally, BST2 acts as a sensor for infection, triggering an antiviral state within the infected cells [1].

Lentiviruses, including HIV-1, have undergone evolutionary adaptations and developed diverse strategies to overcome cellular barriers. Through protein-protein interactions involving viral accessory proteins, lentiviruses often target restriction factors to degradation or sequester them in intracellular compartments. The Vpu (Viral protein U) accessory protein of HIV-1 is one of the strategies developed by the virus to counteract BST2. Vpu reduces the presence of BST2 at the viral budding sites by modifying BST2 intracellular trafficking and degradation. We previously demonstrated that Vpu via its interaction with the autophagy-related protein LC3C subverts a cellular degradation mechanism, resembling LC3-associated pathways, to counteract the antiviral activities of BST2. LC3-associated pathways are processes in which the functions of LC3 isoforms and a subset of autophagy-related (ATG) proteins are defined as non-canonical since outside the context of autophagy. Those processes entail LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). Notably, we found that ATG5 and BECN1 (beclin 1), but not other components of canonical autophagy such as FIP200 and ATG14, act with LC3C in this LC3C-associated degradative pathway induced by HIV-1 infection.

In our latest study, we conducted a detailed examination of the initial events that lead to the targeting of BST2 into an LC3C-associated pathway subverted by Vpu. Additionally, we investigated the relevance of this pathway in Vpu’s ability to counteract the pro-inflammatory signaling triggered by BST2 viral sensing.

Using biochemical approaches, we have demonstrated that ATG5 and BST2 form a complex. This interaction occurs independently of HIV-1 Vpu expression and precedes the recruitment of LC3C by Vpu to the single membrane vesicle of the LC3C-associated pathway. It is important to note that this role of ATG5 is distinct from its involvement in canonical autophagy. Interestingly, the conjugation of ATG12 to ATG5 is unnecessary for the formation of the BST2-ATG5 complex. Furthermore, our findings indicate that a productive HIV-1 infection reduces the formation of ATG5-ATG12 conjugate, making more free ATG5 available, which in turn favors the interaction of unconjugated form of ATG5 with BST2. We also showed that this interaction occurs at the plasma membrane. Depletion of ATG5 results in the accumulation of viral particles clusters at the plasma membrane and blocks the internalization of mature viral particles tethered by BST2. Thus, by forming a complex with BST2, ATG5 targets BST2-tethered viral particles at the plasma membrane.

BST2 through multimerization tether extracellular virions to the cells. BST2 also acts as a viral sensor capable of initiating the recruitment of proteins and signaling cascades. In our study, we have demonstrated that the binding of ATG5 to BST2 is dependent on the integrity of the cytoplasmic tail of BST2 and the cysteine residues implicated in BST2 dimerization and viral particles tethering. Through ATG5 knockdown experiments, we have provided evidence that ATG5 specifically binds to phosphorylated and dimerized BST2 molecules tethering viral particles at the plasma membrane, and directs these viral particles toward the LC3C-associated pathway for subsequent degradation.

BST2 plays a crucial role as a sensor of HIV-1 budding. By tethering viral particles, BST2 activates the NF-kB pathway, which leads to the expression of pro-inflammatory cytokines and establishes an antiviral state within infected cells. Our research highlights the key role of ATG5 in transducing the BST2-mediated signaling triggered by the retention of viral particles. Furthermore, our findings suggest that ATG5 may contribute to BST2-mediated signaling by regulating the recruitment of pSYK (phosphorylated spleen associated tyrosine kinase) to BST2. Additionally, we observed that LC3C and Vpu act downstream of ATG5 to reduce the inflammatory response induced by BST2 viral sensing. Thus, ATG5 is a key mediator in the LC3C-associated pathway exploited by the viral protein Vpu to weaken the host’s immune response to HIV-1 infection.

In conclusion, our study has shed to light into a mechanism in which ATG5 selectively engages the subset of BST2, that tethers and senses the viral particles present at the plasma membrane, through an LC3C-associated pathway. Subsequently, Vpu recruits LC3C to the vesicle containing phosphorylated BST2-tethered viral particles. This recruitment facilitates the rapid docking of this compartment with lysosomes, leading to the degradation of its contents and to the attenuation of the proinflammatory response triggered by BST2 viral sensing (Figure 1). Therefore, the manipulation of the LC3C-associated pathway by Vpu plays a crucial role in regulating the inflammatory response elicited by BST2-mediated viral particles retention. BST2 exhibits broad antiviral activities against various enveloped viruses. Our findings have thus potential wide virological implications, as well as on research on extracellular components trapped by BST2 at the cell surface, such as exosomes or midbodies. Furthermore, the discovery of another novel function of ATG5 different from the one in autophagy, as an adaptor for membrane receptors, presents exciting opportunities for further investigation into the involvement of ATG5 in engaging surface receptors within non-canonical autophagy pathways.

Figure 1.

ATG5 drives the BST2 subpopulation tethering and sensing the virus at the plasma membrane into a LC3C-associated degradative pathway. ATG5 is implicated at the early stage of the internalization of a subset of BST2 molecules at the plasma membrane. ATG5 binds cysteine-linked homodimerized BST2 at the plasma membrane in a Vpu-independent manner, and specifically targets phosphorylated BST2-tethered viral particles into the LC3C-associated pathway. Then, through its interaction with BST2, Vpu recruits LC3C onto the vesicle containing phosphorylated BST2. The LC3C recruitment accelerates the docking of this compartment to the lysosomes and subsequent degradation. ATG5 selectively engages phosphorylated BST2, the specific form that trigger BST2-mediated signaling, into the LC3C-associated pathway. Hence, through targeting of BST2-tethered viral particles for degradation, this LC3C-associated pathway subverted by Vpu attenuates the inflammatory response triggered by BST2-mediated virion retention.

Funding Statement

D.J. holds a fellowship from ANRS and then from SIDACTION. This work is funded by ANRS and SIDACTION.

List of abbreviations

AIDS

acquired immunodeficiency syndrome

ATG5

autophagy related 5

ATG12

autophagy related 12

ATG14

autophagy related 14

BST2/tetherin

bone marrow stromal cell antigen 2

FIP200/RB1CC1

RB1inducible coiled-coil 1

HIV-1

human immunodeficiency virus type 1

MAP1LC3C/LC3C

microtubule associated protein 1 light chain 3 gamma

SYK

spleen associated tyrosine kinase

pSYK

phosphorylated spleen associated tyrosine kinase

Vpu

Viral Protein U

Disclosure statement

No potential conflict of interest was reported by the author(s).