Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

Abstract

Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. Due to their intrinsically low immunogenicity, their ability to infect a variety of tissues in vivo, and their capacity to confer prolonged transgene expression in postmitotic tissues (1), vectors based on adeno-associated virus (AAV) are among the most promising gene therapy tools. Although these properties make AAV an attractive candidate for many clinical applications, some tissues or cell types are not efficiently transduced by AAV vectors, presumably due to the absence of viral receptors, inefficient intracellular trafficking, or viral uncoating (recently reviewed in reference 2).AAVs contain a single-stranded DNA genome, and the entire viral replication cycle-second-strand DNA synthesis, replication of viral genomes, and encapsidation-takes place in the nucleus. Therefore, correct trafficking of incoming virions from the plasma membrane toward the nuclear compartment is of crucial importance for viral or therapeutic gene expression. Following the initial attachment to a primary glycoprotein receptor (heparan sulfate proteoglycan for AAV serotype 2 [AAV2], AAV3, and AAV6; sialic acids for AAV1, AAV4, AAV5, and AAV6; and N-linked galactose for AAV9 [2]), viral particles undergo rapid endocytosis. Whereas more than one e...

Varicella-zoster virus ORF7 interacts with ORF53 and plays a role in its trans-Golgi network localization

“…Firstly, we found that both ORF7 and ORF53 co-localized well with the TGN46 protein ( Figure 3A and 3B), which is widely used as a TGN marker in numerous studies (Barr et al, 2010;Donsante et al, 2011;Zhao et al, 2012;Charles et al, 2014;Nonnenmacher et al, 2015;Pillay et al, 2016). The Pearson's coefficients were 0.70 ± 0.09 and 0.68 ± 0.08 in the co-localized volume of TGN46 with ORF7 and ORF53, respectively (n = 10 in each group).…”

Retrograde Transport from Early Endosomes to the trans -Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

“…However, our studies showed that neither entry nor formation of infectious virus was impaired. In comparison, several families of nonenveloped viruses use retrograde transport for entry (21)(22)(23)(24). The key results, shown by confocal microscopy and CsCl 2 sedimentation, were that depletion of retrograde transport factors inhibited wrapping of MVs and egress.…”

“…Retrograde transport is highly selective and depends on numerous tethering factors, small GTPases, and SNARES (25,26). The small molecule Retro-2 interferes with retrograde transport from early endosome to the TGN specifically and protects against ricin and Shiga-like toxins (27) and virus entry (22)(23)(24), while having little or no apparent cellular toxicity.…”

“…Recycling transmembrane proteins and extracellular molecules that enter cells by endocytosis move in the opposite or retrograde direction from endosomes to the trans-Golgi network. Bacterial (Shiga and cholera) and plant (ricin and abrin) toxins (20) as well as some nonenveloped viruses, including simian virus 40 (SV40), polyomavirus, adeno-associated virus, and papillomavirus (21)(22)(23)(24), exploit the retrograde path to enter cells. Retrograde transport is highly selective and depends on numerous tethering factors, small GTPases, and SNARES (25,26).…”

MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2

“…t-SNARE proteins interact with vesicle SNARE (v-SNARE) proteins and subsequently mediate membrane fusion (45). Several previous reports demonstrated that the SNARE complex is involved in viral infection (46)(47)(48). Meier et al reported that siRNA-mediated knockdown of the SNARE protein VAMP3 resulted in the inhibition of endocytic intracellular trafficking of a bunyavirus, Uukuniemi virus, indicating that SNARE protein-mediated membrane fusion is needed for the efficient entry of Uukuniemi virus into cells (49).…”