title: TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction.
abstract: TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins.
  
  
  OBJECTIVE
  Restriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5α inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5α itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5α is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5α does not require autophagic machinery. These data indicate that the effector functions of TRIM5α can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members.
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Imam
authors.affiliation: Integrative Cell Biology Program, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Talley
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Nelson
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Adarsh
authors.initials: A
authors.lastname: Dharan
authors.affiliation: Department of Biological Sciences, Maryville University, St. Louis, Missouri, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: O'Connor
authors.affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hope
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA Integrative Cell Biology Program, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA ecampbell@luc.edu.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Campbell
mesh_terms: D000818:Animals; D000090103:Antiviral Restriction Factors; D016645:Aotidae; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000235:genetics*; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008253:Macaca mulatta; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D059748:Proteolysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics / Q000254:growth & development*; D012192:Retroviridae Infections / Q000276:immunology / Q000821:virology*; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases; D014758:Viral Core Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*; D059666:Lysosomal-Associated Membrane Protein 3
chemical_list: D000090103:Antiviral Restriction Factors; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D002352:Carrier Proteins; D051907:Lysosomal Membrane Proteins; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases; D014758:Viral Core Proteins; C502778:ATG5 protein, human; C491997:BECN1 protein, human; C114233:LAMP3 protein, human; D059666:Lysosomal-Associated Membrane Protein 3; C502070:MAP1LC3B protein, human; C118585:P62 protein, human; C040929:bafilomycin A1; C477346:TRIM5 protein, human