title: Functional evidence for the involvement of microtubules and dynein motor complexes in TRIM5α-mediated restriction of retroviruses.
abstract: The tripartite motif (TRIM) family of proteins includes the TRIM5α antiretroviral restriction factor. TRIM5α from many Old World and some New World monkeys can restrict the human immunodeficiency virus type 1 (HIV-1), while human TRIM5α restricts N-tropic murine leukemia virus (N-MLV). TRIM5α forms highly dynamic cytoplasmic bodies (CBs) that associate with and translocate on microtubules. However, the functional involvement of microtubules or other cytoskeleton-associated factors in the viral restriction process had not been shown. Here, we demonstrate the dependency of TRIM5α-mediated restriction on microtubule-mediated transport. Pharmacological disruption of the microtubule network using nocodazole or disabling it using paclitaxel (originally named taxol) decreased the restriction of N-MLV and HIV-1 by human or simian alleles of TRIM5α, respectively. In addition, pharmacological inhibition of dynein motor complexes using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and small interfering RNA-mediated depletion of the dynein heavy chain (DHC) similarly decreased TRIM5α-mediated restriction. The loss in restriction resulting from either the disassembly of microtubules or the disruption of dynein motor activity was seen for both endogenous and overexpressed TRIM5α and was not due to differences in protein stability or cell viability. Both nocodazole treatment and DHC depletion interfered with the dynamics of TRIM5α CBs, increasing their size and altering their intracellular localization. In addition, nocodazole, paclitaxel, and DHC depletion were all found to increase the stability of HIV-1 cores in infected cells, providing an alternative explanation for the decreased restriction. In conclusion, association with microtubules and the translocation activity of dynein motor complexes are required to achieve efficient restriction by TRIM5α.
  
  
  OBJECTIVE
  The primate innate cellular defenses against infection by retroviruses include a protein named TRIM5α, belonging to the family of restriction factors. TRIM5α is present in the cytoplasm, where it can intercept incoming retroviruses shortly after their entry. How TRIM5α manages to be present at the appropriate subcytoplasmic location to interact with its target is unknown. We hypothesized that TRIM5α, either as a soluble protein or a high-molecular-weight complex (the cytoplasmic body), is transported within the cytoplasm by a molecular motor called the dynein complex, which is known to interact with and move along microtubules. Our results show that destructuring microtubules or crippling their function decreased the capacity of human or simian TRIM5α to restrict their retroviral targets. Inhibiting dynein motor activity, or reducing the expression of a key component of this complex, similarly affected TRIM5α-mediated restriction. Thus, we have identified specific cytoskeleton structures involved in innate antiretroviral defenses.
authors.affiliation: Laboratory of Retrovirology, Department of Medical Biology and BioMed Group, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.
authors.forename: Paulina
authors.initials: P
authors.lastname: Pawlica
authors.forename: Valerie
authors.initials: V
authors.lastname: Le Sage
authors.forename: Nolwenn
authors.initials: N
authors.lastname: Poccardi
authors.forename: Michel J
authors.initials: MJ
authors.lastname: Tremblay
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Mouland
authors.forename: Lionel
authors.initials: L
authors.lastname: Berthoux
mesh_terms: D000818:Animals; D000090103:Antiviral Restriction Factors; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004398:Dyneins / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D009052:Leukemia Virus, Murine / Q000276:immunology*; D008253:Macaca mulatta; D008870:Microtubules / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases
chemical_list: D000090103:Antiviral Restriction Factors; D002352:Carrier Proteins; D011506:Proteins; D000070584:Tripartite Motif Proteins; C477346:TRIM5 protein, human; C482587:TRIM5(alpha) protein, rhesus monkey; D044767:Ubiquitin-Protein Ligases; D004398:Dyneins