Regulation of Autophagosome–Lysosome Fusion by Human Viral Infections

Autophagy plays a fundamental role in maintaining cellular homeostasis by eliminating intracellular components via lysosomes. Successful degradation through autophagy relies on the fusion of autophagosomes to lysosomes, which leads to the formation of autolysosomes containing acidic proteases that degrade the sequestered materials. Viral infections can exploit autophagy in infected cells to balance virus–host cell interactions by degrading the invading virus or promoting viral growth. In recent years, cumulative studies have indicated that viral infections may interfere with the fusion of autophagosomes and lysosomes, thus benefiting viral replication and associated pathogenesis. In this review, I provide an overview of the current understanding of the molecular mechanism by which human viral infections deregulate autophagosome–lysosome fusion and summarize the physiological significance in the virus life cycle and host cell damage.


Introduction
Autophagy is a stress-response pathway in which cytosolic components are sequestered within double membrane-bound autophagosomes, which fuse with lysosomes to form autolysosomes containing lysosomal proteases to degrade the engulfed materials [1][2][3].Upon virus infection, host cellular autophagy can be induced to eliminate the infecting virus, thus serving as the first line of antiviral response to restrict virus growth [4][5][6][7].Some viruses, such as herpesviruses, can suppress autophagy initiation in infected cells to counteract this host defense mechanism [8,9].In some cases of viral infection, such as in poliovirus and coronavirus infection [10][11][12][13][14], autophagy induction by infected cells promotes the generation of double-membrane vesicles to enhance viral replication.In addition, the activation of autophagy by several viruses, including flaviviruses and enteroviruses, can facilitate virus spread through the assembly and release of infectious particles through autophagic vacuoles [15][16][17][18][19][20].
Moreover, viruses, such as hepatitis C virus (HCV) and influenza A virus (IAV), can subvert autophagy to repress innate antiviral immunity and promote viral growth [21][22][23][24][25].Recently, accumulated evidence has demonstrated that virus infection interferes with the fusion of autophagosomes and lysosomes and inhibits the acidification of lysosomes to enhance the replication of viruses and the maturation of viral particles.In this review, I outline the current knowledge on how human viruses regulate autophagosome-lysosome fusion to promote the viral life cycle.Then, the underlying molecular mechanisms are reviewed.Finally, the physiological significance of the ability of viruses to modulate the fusion of autophagosomes and lysosomes on the pathogenicity of virus-related human diseases is discussed.
The minus end-directed and retrograde movement of microtubules allows autophagosomes to meet lysosomes in the perinuclear region, thus facilitating their fusion.This transport of autophagosomes is mediated by a Rab7-interacting protein (RILP) and its association with the dynein-dynactin motor complex of microtubules and interaction with LC3 on autophagosomes (Figure 2) [91,100,101].In addition, RILP also interacts with a cholesterol sensor protein, oxysterol-binding protein (OSBP)-related protein 1L (ORP1L), to promote the minus end-directed movement of autophagosomes on microtubules (Figure 2) [102].In addition, ORP1L also cooperates with RILP to recruit the HOPS complex via PLEKHM1 to promote autophagosome-lysosome fusion.In addition, FYVE and coiled-coil (CC) domain-containing protein (FYCO1), another Rab7 effector, can form an adaptor complex with Rab7 and LC3B to promote plus end-directed transport of autoph- The minus end-directed and retrograde movement of microtubules allows autophagosomes to meet lysosomes in the perinuclear region, thus facilitating their fusion.This transport of autophagosomes is mediated by a Rab7-interacting protein (RILP) and its association with the dynein-dynactin motor complex of microtubules and interaction with LC3 on autophagosomes (Figure 2) [91,100,101].In addition, RILP also interacts with a cholesterol sensor protein, oxysterol-binding protein (OSBP)-related protein 1L (ORP1L), to promote the minus end-directed movement of autophagosomes on microtubules (Figure 2) [102].In addition, ORP1L also cooperates with RILP to recruit the HOPS complex via PLEKHM1 to promote autophagosome-lysosome fusion.In addition, FYVE and coiled-coil (CC) domain-containing protein (FYCO1), another Rab7 effector, can form an adaptor complex with Rab7 and LC3B to promote plus end-directed transport of autophagosomes on microtubules, presumably through the motor protein kinesin (Figure 2) [90].Histone deacetylase-6 (HDAC6)-recruited cortactin and its consequent F-actin polymerization also participate in the fusion of autophagosomes to lysosomes [121].
In addition to Rab7, several Rab family proteins diversely regulate autophagosomelysosome fusion (Figure 2).Rab34B and its GTPase-associated protein OATL1 (also named TBC1D25) suppress the fusion of autophagosomes to lysosomes [122], while Rab21 and Rab24 promote the fusion of autophagosomes and lysosomes via their interactions with RILP and VAMP8 [123][124][125].Furthermore, the UV-irradiation-resistance-associated gene (UVRAG), which is a component of the PI3KC3 complex shown to interact with Beclin 1 [32], promotes the fusion of autophagosomes with lysosomes by enhancing Rab7 activity and facilitating the recruitment of GRASP55 [36,126,127].In contrast, another component of the PI3KC3 complex, the RUN domain and cysteine-rich domain-containing Beclin 1-interacting protein (Rubicon), negatively regulates autophagy by suppressing autophagosome-lysosome fusion [36,120,127].

Hepatitis C Virus
HCV is a positive, single-stranded RNA and enveloped virus that belongs to the genus Hepacivirus within the Flaviviridae family [131][132][133][134]. Chronic HCV infection often leads to the development of end-stage liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC) [131,132].In the late 2000s, Sir et al. first demonstrated that the replication of HCV RNA (JFH1 strain, belonging to genotype 2a) induces the accumulation of autophagosomes in the human hepatoma cell line Huh7 (Table 1) [135].Additionally, bafilomycin-A1 (BAF-A1) treatment fails to further increase the level of LC3-II, which is a hallmark of autophagosome formation, in HCV-infected cells compared to that in mockinfected cells, suggesting that HCV infection may block autophagic flux and interfere with the fusion of autophagosomes to lysosomes [135].HCV-induced incomplete autophagy was required to efficiently replicate HCV viral RNA [135].Later, Sir and colleagues showed that HCV RNA colocalizes with GFP-LC3 puncta-and endogenous LC3 puncta-labeled autophagosomes in Huh7 cells harboring a subgenomic replicon, which contains an HCV genome fragment of nonstructural (NS) 3 to 5B (Table 1) [136].Additionally, HCV NS5A and NS5B function in the assembly of the HCV RNA replication complex, which was detected in endogenous LC3 puncta in HCV replicon cells [136].Moreover, HCV RNA, NS5A, and NS5B are found in purified autophagosomal membranes in HCV replicon cells, supporting the notion that HCV induces the formation of autophagosomes to support the formation of the membranous compartment for the replication of viral RNA [136].
On the other hand, Wang et al. reported that HCV infection induces the protein expression of Rubicon earlier than UVRAG infection (Table 1) [137].Genetic knockdown of Rubicon promotes the formation of RFP + /GFP − -labeled autolysosomes of the mRFP-GFP-LC3 reporter in HCV-infected cells and suppresses HCV viral replication [137].In addition, overexpression of Rubicon delays RFP + /GFP − -labeled autolysosome maturation and promotes HCV viral replication in HCV-infected cells [137].In contrast, ectopic expression of UVRAG in HCV-infected cells facilitates the formation of RFP + /GFP − autolysosomes and enhances the replication of HCV viral RNA [137].Moreover, the authors showed that HCV NS4B alone can induce Rubicon expression and promote autophagosome maturation [137].These studies point out that HCV infection may transiently inhibit the fusion of autophagosomes to lysosomes via NS4B-mediated upregulated expression of the Rubicon protein and the suppression of UVRAG protein expression [137].Along with this study, HCV infection-induced Rubicon expression was reported to repress autophagy and activate innate immune response [138].Another study by Weinman's group showed that HCV infection upregulates the protein expression of ADP-ribosylation factor-like protein 8B (Arl8b), which is an Arf-like GTPase required for the trafficking of lysosomes, leading to the redistribution of lysosomes to the peripheral region and the repression of autophagic flux (Table 1) [139].Gene silencing of Arl8b in HCV-infected cells restores autophagic flux and suppresses the release of infectious virions [139].These results suggest that HCV can activate Arf8b expression to block the fusion between autophagosomes and lysosomes.

Enteroviruses
Enterovirus (EV) is an unenveloped, positive-sense, single-stranded RNA virus belonging to the Picornaviridae family [140][141][142][143].Most EV infections are asymptomatic and selflimiting but still cause severe disease manifestations, such as hand-foot-and-mouth disease and neurological disorders in infants, children, and immunodeficient persons [140][141][142][143]. Coxsackievirus B3 (CVB3) belongs to the Enterovirus genus within the Picornaviridae family and often leads to gastrointestinal distress and cardiomyopathy in infected individuals [144].CVB3 infection interferes with the fusion of autophagosomes with lysosomes in infected cells [145].Luo's group showed that CVB3 infection induces the accumulation of GFP-LC3-labeled autophagosomes in the heart tissues of infected GFP-LC3 transgenic mice (Table 1) [145].Also, CVB3 infection into HEK293 human embryonic kidney cells leads to the predominant expression of RFP + /GFP + -labeled autophagosomes of the mRFP-GFP-LC3 reporter, suggesting that CVB3 infection represses autophagic flux [145].Moreover, the authors demonstrated that CVB3 proteinase 3C induces the cleavage of SNAP29 and PLEKHM1 at glutamine (Q) 161 and Q668, respectively, in infected cells [145].In addition, CVB3 infection blocks the interaction between STX17 and VAMP8 [145].Gene silencing of SNAP29 and PLEKHM1 in CVB3-infected HeLa human cervical cancer cells suppresses autophagic flux and promotes virus replication [145].These studies indicate that CVB3 infection may inhibit the assembly of the STX17-SNAP29-VAMP8 SNARE complex and the recruitment of membrane tethers by proteinase 3C-cleaved SNAP29 and PLEKHM1, thus blocking the fusion between autophagosomes and lysosomes [145].
Meanwhile, Tian and colleagues reported that CVB3 infection promotes the formation of autophagosomes in HeLa cells in a dose-dependent manner (Table 1) [146].Additionally, BAF-A1 treatment did not further increase the LC3-II level in CVB3-infected cells, suggesting that CVB3 infection inhibits autophagic flux [146].CVB3 infection also leads to the accumulation of RFP + /GFP + -labeled autophagosomes and a decrease in the mRNA level of STX17 in infected cells [146].Moreover, ectopic expression of STX7 in CVB3-infected cells results in the efficient fusion of autophagosomes with lysosomes and prevents viralinduced apoptosis of infected cells [146].These results suggest that CVB3 may interfere with autophagosome-lysosome fusion by suppressing the gene expression of STX17 [146].Similarly, enterovirus D68 (EV-D68), which is another EV that predominantly causes acute flaccid myelitis in infected children, has also been shown to subvert the fusion of autophagosomes and lysosomes (Table 1) [147].EV-D68 infection in HeLa cells increases the expression of LC3-II and induces the cleavage of sequestosome 1 (SQSTM1), which is a cargo receptor that targets degradative cargoes for autophagic degradation [147].Also, EV-D68 induces the cleavage of SNAP29 at Q161 by the 3C protease but also enhances the protein expression of STX17 in infected cells [147].Genetic knockdown of SNAP29 inhibits virus replication and virion secretion at the early stage of infection [147].Moreover, these authors identified another SNAP protein, SNAP47, which may analogously regulate the autophagic process in EV-D68-infected cells and promote the production of infectious intracellular viral particles [147].These studies imply that EV-D68 may block the fusion of autophagosomes to lysosomes in a fashion similar to that of CVB3 [147].

Influenza A Virus
IAV is an enveloped negative-sense single-strand RNA virus belonging to the Alphainfluenzavirus genus within the Orthomyxoviridae family [148][149][150].IAV infection leads to systematic symptoms ranging from respiratory disease symptoms, such as fever, cough, and sore throat, to muscle pain, heart failure, and lethal pneumonia [148][149][150].Gannage and colleagues first demonstrated that IAV infection in A549 human lung epithelial cells induces the accumulation of GFP-LC3-labeled autophagosomes, which colocalize with polyubiquitinated compartments and SQSTM1 (Table 1) [151].Additionally, IAV-triggered autophagosomes do not fuse with lysosomes in infected cells [151].In addition, the authors showed that IAV matrix protein 2 (M2) alone can induce autophagosome accumulation by blocking the fusion of autophagosomes to lysosomes [151].Interference with autophagy in IAV-infected cells promotes cell apoptosis and the extracellular release of viral proteins [151].These studies point out that the IAV M2 protein may repress autophagosome-lysosome fusion [151].In line with these findings, another study by Chanda's group showed that IAV M2 interacts with TBC1D5/OATL1 through its cytoplasmic tail in infected HEK293T cells (Table 1) [152].The binding of IAV M2 to TBC1D5/OATL1 promotes the targeting of IAV M2 to lysosomes, and IAV M2 interferes with the interaction between Rab7 and TBC1D5/OATL1 in infected HEK293T cells [152].Genetic silencing of TBC1D5/OATL1 in IAV-infected A549 cells promotes virus replication and promotes virus-induced lethality in infected mice [152], suggesting that TBC1D5/OATL1 restricts IAV infection [152].These studies indicate that IAV M2 may block the fusion of autophagosomes to lysosomes by binding to TBC1D5/OATL1 and interrupting the interaction between TBC1D5/OATL1 and Rab7 [152].
Meanwhile, Zhang et al. reported that among thirteen SARS-CoV-2 viral genomeencoded proteins, ectopic expression of ORF3a induces elevations in LC3-II and SQSTM1 protein expression in HEK293T cells (Table 1) [157].The exogenous expression of SARS-CoV-2 ORF3a in HeLa and A549 cells similarly leads to upregulated levels of LC3-II and SQSTM1 in a dose-dependent manner, and ORF3a overexpression triggers the accumulation of GFP-LC3-labeled puncta and RFP + /GFP + autophagosomes of a mCherry-GFP-LC3 reporter [157].In addition, the authors showed that SARS-CoV-2 ORF3a localizes to LAMP1-labeled lysosomes in HeLa cells and that the transmembrane (TM) domain and C-terminus of SARS-CoV-2 ORF3a are both required for the induction of autophagosome accumulation [157].Another study indicated that SARS-CoV-2 ORF3a directly binds to VPS39 via tyrosine (Y) 160 at its C-terminal region in HeLa cells, thus interrupting the interaction between VPS39 and Rab7 and coincidently disrupting the association of SNAP29 with VAMP8, which is necessary for the assembly of the SNARE complex [157].These studies imply that SARS-CoV-2 ORF3a inhibits autophagosome-lysosome fusion by interacting with VPS39 and interfering with the formation of functional fusion machinery [157].
Similarly, Qu and colleagues showed that in a similar fashion to ORF3a overexpression, SARS-CoV-2 infection leads to an increased amount of LC3-II and SQSTM1 in HeLa-hACE2 cells, which support a complete SARS-CoV-2 life cycle and VERO-E6 African green monkey kidney cells (Table 1) [158].The authors demonstrated that SARS-CoV-2 ORF3a specifically binds to UVRAG and interferes with the interaction between UVRAG and the PI3KC3 complex [158].Genetic depletion of ATG3 and ATG5 dramatically inhibits the replication of SARS-CoV-2 viral RNA in infected mouse embryonic fibroblast (MEF)-hACE2 cells, while SARS-CoV-2 ORF3a overexpression enhances viral RNA replication in SARS-CoV-2-infected Calu-3 human lung adenocarcinoma cells [158].These studies suggest that SARS-CoV-2 ORF3a targets UVRAG, thus preventing its interaction with the PI3KC3 complex and thereby repressing the fusion of autophagosomes with lysosomes [158].Notably, SARS-CoV ORF3a is not able to inhibit autophagosomelysosome fusion [157,158].Recently, Zhu et al. showed that adeno-associated virus (AAV) gene delivery of SARS-CoV-2 ORF3a in mouse brains leads to neurological disturbance, neurodegeneration, and cell death (Table 1) [159].In addition, SARS-CoV-2 ORF3a expression also induces the glial response and activates inflammatory gene expression in the brains of mice [159].Moreover, the authors reported that ectopic expression of SARS-CoV-2 ORF3a in HeLa cells blocks autophagosome-lysosome fusion and also impairs the degradation of glycosphingolipid, suggesting that the deregulation of autolysosome maturation by SARS-CoV-2 ORF3a may interfere with sphingolipid homeostasis [159].

Human Parainfluenza Virus
Human parainfluenza virus (HPIV) is an enveloped, negative-sense, single-stranded RNA virus belonging to the Paramyxoviridae family [161][162][163].HPIV infection often causes respiratory diseases, including colds, croup, and bronchiolitis, in children and adults; in some severe cases, HPIV can ultimately progress to pneumonia [161][162][163].Ding et al. demonstrated that HPIV3 infection in LLC-MK-2 (MK-2) monkey kidney cells and HeLa cells increases LC3-II protein expression and the number of GFP-LC3 puncta in which lysosomes do not exist (Table 1) [164].Chloroquine (CQ) and BAF-A1, which are inhibitors of autolysosome maturation, did not increase the level of LC3 in HPIV3-infected MK-2 cells [164].HPIV3 leads to the accumulation of RFP + /GFP + -labeled autophagosomes of the mCherry-GFP-LC3 reporter in infected MK-2 cells [164].Interference with autophagosome formation by 3-MA and gene knockout of ATG7 inhibits the production of extracellular virions in infected MK-2 and MEF cells [164].Among the viral proteins of HPIV3, ectopic expression of phosphoprotein (P) protein alone is sufficient to induce LC3-II upregulation, the colocalization of GFP-LC3 puncta with lysosomes, and the accumulation of RFP + /GFP + autophagosomes of the mCherry-GFP-LC3 reporter in HeLa cells [164].Further analysis revealed that the HPIV3 P protein directly binds to SNAP29 via the N-terminal region [164].This interaction prevents the association of SNAP29 with STX17 rather than with VAMP8 [164].Deletion of the N-terminal region required for SNAP29 binding in the HPIV3 P protein inhibits HPIV3 P-induced autophagosome accumulation [164].These studies suggest that the HPIV3 P protein inhibits autophagosome-lysosome fusion by interacting with SNAP29, thus promoting the accumulation of autophagosomes to produce infectious viruses [164].

Other Viruses
In addition to the viruses mentioned above, other viruses, such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), have been shown to modulate the fusion of autophagosomes with lysosomes [165,166].In EBV-producing cells, induced autophagosomes do not colocalize with lysosomes, which coincides with the downregulation of Rab7 protein expression, thus preventing the degradation of EBV and enhancing virus replication (Table 1) [165].Similarly, the activation of the KSHV lytic cycle induces the formation of autophagosomes, which cannot colocalize with lysosomes [166].A reduction in Rab7 also mediates this blockade of autophagosome-lysosome fusion via KSHV lytic cycle activation [166].These studies indicate that oncogenic viruses may deregulate the fusion of autophagosomes to lysosomes, thus promoting their escape from autophagic degradation and switching the latent-lytic infection cycle.

Virus
Effects on the Fusion of Autophagosomes with Lysosomes References HCV 1. Replication of viral RNA activates incomplete autophagy.2. HCV viral RNA replication inhibits autophagic flux and induces autophagosome accumulation.
3. The viral-mediated inhibition of autophagy promotes virus replication.

Conclusions and Perspectives
In recent years, several viruses have been shown to inhibit the fusion of autophagosomes with lysosomes, thus resulting in the accumulation of autophagosomes in which viruses grow.Most of these viruses interrupt the assembly of the SNARE complex via protein-mediated competitive binding, the downregulation of expression, and the proteolysis of molecules involved in autophagosome-lysosome fusion.So far, the detailed mechanism underlying this regulation remains debated and needs further investigation.For example, the spatiotemporal control of the timely actions of viral proteins in the fusion process is unclear.Notably, whether these viruses interfere with the fusion of autophagosomes to lysosomes is still debated, at least for HCV, as several studies have shown that viralinduced autophagy progresses to autolysosome maturation and is used for the turnover of organelles [21,167,168].Little is known about how infected cells deal with the large population of autophagosomes when viruses repress their fusion to lysosomes, and whether this deregulation of fusion contributes to viral-induced pathogenesis via the dysfunction of autophagic degradation is also unclear.Most importantly, whether and how viruses interfere with autophagosome-lysosome fusion in physiologically relevant cell contexts, such as in small animal models of infection and in the tissues of individuals infected with viruses, remains largely unknown, as most past studies have used cell culture infection as a research model.In perspective, further investigations are needed to elucidate the molecular mechanism by which viruses block the fusion of autophagosomes with lysosomes and to explore the physiological significance of the dysregulation of autophagosome-lysosome fusion in virus-associated diseases in humans.

Figure 1 .
Figure 1.Schematic diagram of the autophagy process.Stresses, such as starvation, inhibit the activity of mTOR, thus promoting ER translocation of the ULK complex to recruit the PI3KC3 complex.The activated PI3KC3 complex generates PtdIns(3)P to recruit DFCP1 and WIPIs for isolation membrane (IM) nucleation.Elongation of the IM/phagophore to a closed autophagosome involves

Figure 1 .
Figure 1.Schematic diagram of the autophagy process.Stresses, such as starvation, inhibit the activity of mTOR, thus promoting ER translocation of the ULK complex to recruit the PI3KC3 complex.The activated PI3KC3 complex generates PtdIns(3)P to recruit DFCP1 and WIPIs for isolation membrane (IM) nucleation.Elongation of the IM/phagophore to a closed autophagosome involves the ubiquitin-like conjugation system for the formation of ATG12-ATG12-ATG16L and ATG8/LC3s-PE conjugates.The autophagosome subsequently fuses with a lysosome to form an autolysosome, in which the cytosolic components are degraded.The SNARE complex, tethers, Rab family proteins, and cytoskeletal motors facilitate autophagosome-lysosome fusion.When autophagy is terminated, the tubular proto-lysosomes emerge from autolysosomes, promoting the regeneration of lysosomes.

Pathogens 2024 , 17 Figure 2 .
Figure 2. Specific molecules function in the fusion of autophagosomes with lysosomes.Various functional molecules, including the SNARE complex, tethering factors, Rab family proteins, cytoskeletal motors, autophagosomal proteins, phosphoinositides, and adaptor proteins, are required for autophagosome-lysosome fusion.

Figure 2 .
Figure 2. Specific molecules function in the fusion of autophagosomes with lysosomes.Various functional molecules, including the SNARE complex, tethering factors, Rab family proteins, cytoskeletal motors, autophagosomal proteins, phosphoinositides, and adaptor proteins, are required for autophagosome-lysosome fusion.

Table 1 .
Regulation of autophagosome-lysosome fusion by human viruses.