Honglin Luo

Lung cancer is the leading cause of cancer-related death for both sexes worldwide. KRAS and TP53/RB1 are the most frequently mutated genes in lung adenocarcinoma and small cell lung cancer, respectively. However, lung cancer associated with these gene mutations has a poor outcome and there is a lack of effective treatment options. Recent advances in virus-based cancer treatment, termed virotherapy, provide a promising new treatment option. Oncolytic viruses are a group of viruses that are either naturally capable of or can be genetically engineered to specifically destroy cancer cells while sparing normal cells. My laboratory previously found that coxsackievirus B3 (CVB3) has extremely potent oncolytic effects against KRAS-mutant lung adenocarcinoma. Nevertheless, the evident toxicity restricts its use for cancer therapy. In this dissertation, I aimed to engineer CVB3 to decrease its damage to normal tissues. My hypothesis was that modification of CVB3 by inserting target-sequence (TS) of tumor-suppressive and/or organ-selective miRNA will reduce its toxicity, while retaining oncolytic potency. I generated a miRNA-modified CVB3 by inserting tumor-suppressive miR-145/-143-TS into the 5’UTR of viral genome. In vitro experiments revealed that this miR-CVB3 strongly infects and lyses both KRAS- and TP53/RB1-mutant lung cancer cells, but with a markedly reduced cytotoxicity toward normal cells. In vivo study using a xenograft mouse model demonstrated that a single dose of the miR-CVB3 via systemic administration significantly suppresses tumor growth with greatly attenuated viral pathogenesis as compared to wildtype CVB3. Notably, after a prolonged treatment (>35 days), reversion mutants (loss of miRNA-TS inserts) were identified in ~40% mice, revealing the instability of miR-CVB3. To improve the stability and further reduce the toxicity, I re-engineered CVB3 by replacing the same length of viral genome at the non-coding region with TS of cardiac-selective miR-1/miR-133 and pancreas-enriched miR-216/miR-375 or inserting these miRNA-TS into the coding P1 region of viral genome. Serial passaging of these newly established CVB3s in cultured cells validated significantly improved stability compared with the initial miR-CVB3. Their safety was also verified in immunocompetent and tumor-bearing immunodeficient mice. Taken together, my research provides valid strategies to develop CVB3 as a safe oncolytic virus for lung cancer treatment.

View record

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons in the central nervous system. Emerging evidence suggests that viruses such as enterovirus (EV) might play a role during ALS pathogenesis. However, available clinical data about EV association with ALS are inconclusive, and the underlying mechanisms remain unclear. EV is a group of highly neurotropic RNA viruses that include poliovirus and non-polioviruses (e.g., coxsackievirus). Since the near eradication of poliovirus, there are emerging reports of severe neurological complications associated with non-polioviruses. The objective of this dissertation is to understand the potential role of EV in ALS pathogenesis. We recently discovered that EV infection in vitro led to ALS-like pathologies such as TDP-43 cleavage, mislocalization, and aggregation. I hypothesize that EV infection could induce and exacerbate ALS-like phenotypes through EV-induced neurotoxicity and ALS-related pathologies (e.g., TDP-43 mislocalization). To address this hypothesis, I demonstrated that infection with coxsackievirus B3 (CVB3), an EV model in this dissertation, in the brain of Balb/c mice resulted in tissue damage, immune cell infiltration, and TDP-43 mislocalization. I further showed that CVB3 infection exacerbated motor dysfunctions and reduced mouse lifespan in SOD1G85R mice (an ALS mouse model that develops progressive ALS-like phenotypes), but not in non-transgenic normal mice, suggesting that CVB3 infection is likely a risk factor but not a cause for ALS. I then discovered that early but not late treatment with ribavirin, a nucleoside analog anti-RNA viral drug, mitigated CVB3-mediated disease exacerbation in SOD1G85R mice, suggesting a potential involvement of “prion-like mechanism” independent of persistent infection in the development of ALS. Finally, I investigated possible additional mechanisms, other than TDP-43 pathology, neuroinflammation, and damage, related to EV-induced neurotoxicity and ALS-related pathology. I reported that FUS (an ALS-associated RNA-binding protein) plays an anti-viral role against CVB3 by interfering with virus translation and through promoting type I interferon and proinflammatory cytokine/chemokine gene expression. Collectively, I showed that CVB3 infection acts as a risk factor for ALS, with multiple potential contributing mechanisms. The findings under its current setup are significant because they provide first-of-its-kind in vivo evidence supporting EV as a risk factor for ALS.

View record

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus of the enterovirus genus that is implicated in diverse human pathologies from viral myocarditis to neurological disorders. CVB3 infection has been linked to the cellular activation of the host autophagy pathway but the underlying mechanisms remain unclear. The central hypothesis of the current dissertation is that that CVB3 usurps the host autophagy pathway to promote viral propagation. To address this hypothesis, I proposed to investigate how CVB3 disrupts important steps in the autophagy pathway including the initiation of autophagosome biogenesis, selective recruitment of cargo, fusion of autophagosomes with lysosomes, and overall lysosomal function. I have demonstrated that CVB3 infection disrupts multiple stages of the autophagy pathway to favor viral pathogenesis, including the initiation of autophagosome biogenesis, the selective recruitment of substrates, the fusion of autophagosomes with lysosomes, as well as lysosomal function. In particular, I uncovered that CVB3 targets autophagy initiation factors such as ULK1/2, through viral proteinase-mediated cleavage, to disrupt canonical autophagy signaling that is activated following physiological stimuli such as starvation. Instead, I showed that CVB3 utilizes viral proteins to initiate non-canonical autophagy that relies on PI4PKIIIβ kinase. In addition to disrupted autophagosome biogenesis, I reported that CVB3 also targets the selective autophagy process by cleaving autophagy adaptor proteins NDP52/CALCOCO2 through the activity of viral proteinase 3C, consistent with our previous reports on adaptor proteins SQSTM1/p62 and NBR1. Furthermore, I uncovered that CVB3 impairs the clearance function of autophagy by disrupting the autophagosome-lysosome fusion process, in part through the cleavage of fusion adaptor and tethering factors SNAP29 and PLEKHM1, respectively. Lastly, I identified that CVB3 targets the master regulator of lysosomal biology, TFEB, through viral proteinase 3C-mediated cleavage to disrupt lysosome function. Collectively, I identify viral proteinases as important pathogenic factors that not only facilitate viral maturation but also disrupt the cellular recycling machinery of autophagy at multiple stages. These findings are significant because they provide a strong foundation for targeting autophagy as a strategy to combat viral pathogenesis.

View record

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite a better understanding of the molecular mechanisms of lung cancer and the subsequent emergence of targeted therapies, treatment responses are typically short-lived. Oncolytic virotherapy provides a possible alternative direction for controlling this incurable disease. Coxsackievirus type B3 (CVB3) is a common human pathogen associated with viral myocarditis in young adults. Due to its highly lytic nature and ability to selectively replicate within cancerous cells, I hypothesize that CVB3 can be developed as an oncolytic virus. Here we demonstrated that in vitro, CVB3 specifically targets KRAS-mutant (KRASmut) non-small-cell lung cancer (NSCLC), a subtype of NSCLC with limited treatment options. Furthermore, we showed in vivo that intratumoral injection of CVB3 significantly reduces tumor volumes in patient-derived KRASmut NSCLC xenograft models. Mechanistically, we found that aberrant activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and elevated expression of the coxsackievirus and adenovirus receptor (CAR), the primary receptor for CVB3 internalization, are associated with preferential replication of CVB3 within KRASmut NSCLC. However, despite a satisfactory tumor regression rate, CVB3 treatment leads to the onset of viral myocarditis in immunocompromised mouse models, indicating that potential safety issues need to be addressed prior to its potential application in lung cancer therapy. It is known that CVB3 subverts host machinery to gain survival advantages, and this process is highly associated with a spectrum of human disorders. We reported that Grb2-associated binding protein 1 (GAB1), a scaffolding adaptor protein responsible for intracellular signaling assembly and transduction, plays a crucial role in regulating compensatory cardiac response to aging and hemodynamic stress. Furthermore, we demonstrated that both GAB1 and Grb2-associated binding protein 2 (GAB2, a functional homologue of GAB1), are proteolytically cleaved after CVB3 infection by virus-encoded protease 2Apro, independent of caspase activation. We showed that virus-induced cleavage of GAB1 is beneficial for viral growth as the resulting cleavage fragment (GAB1-N₁₋₁₇₄) further enhances ERK1/2 activation and promotes viral replication. Taken together, our findings suggest that CVB3 is a potent oncolytic agent against KRASmut NSCLC, and that elimination of CVB3-induced cardiotoxicity would significantly enhance the safety of this virotherapy.

View record

Viral infection affects a multitude of cellular processes to facilitate successful replication. Such responses include the formation of stress granules (SGs) and the activation of autophagy. SGs are stalled translational complexes and function to restore cellular homeostasis after stress. Autophagy is a cellular process that recycles misfolded proteins and damaged organelles and plays an important role in various stress responses. We previously demonstrated that infection with Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, disrupts the autophagic process to support effective viral replication. However, the interplay between CVB3 and SGs, and the ability of SGs to regulate autophagy have not been investigated. Here we showed that SGs are formed early and actively disassembled late during CVB3 infection due to viral protease 3Cpro-mediated cleavage of Ras-GAP SH3 domain binding protein 1 (G3BP1), a key nucleating protein of SGs. Overexpression of G3BP1 inhibits CVB3 replication, indicating an anti-viral function of SGs. We further demonstrated that the C-terminal product of G3BP1 has a toxic gain-of-function that further inhibits SG formation. We also examined the interaction between CVB3 and the transactive response DNA-binding protein-43 (TDP-43), an RNA binding protein that mislocates to SGs under cellular stress. We found that TDP-43 is translocated from the nucleus to SGs upon infection through the activity of viral protease 2Apro, followed by cleavage by protease 3Cpro. The C-terminal product of TDP-43 is quickly degraded by the proteasome, whereas the N-terminal truncate acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Knockdown of TDP-43 results in an increase in viral titres, suggesting a protective role for TDP-43 in CVB3 infection. Lastly, we explored the possible role of G3BP1-SGs in regulating autophagy. We showed that G3BP1 inhibits autophagic flux, likely by binding to cytoplasmic signal transducer and activator of transcription 3 (STAT3). Taken together, our results reveal that the host SGs and associated proteins, including G3BP1 and TDP-43, are utilized and modified during CVB3 infection to promote efficient viral replication and induce viral pathogenesis. Moreover, we propose a novel mechanism by which G3BP1 binds cytoplasmic STAT3 to inhibit autophagy.

View record

Coxsackievirus infection induces an abnormal accumulation of ubiquitin aggregates that are generally believed to be harmful to the cells and play a key role in the pathogenesis of coxsackievirus-induced myocarditis and dilated cardiomyopathy. Selective autophagy mediated by autophagy adaptor proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), is an important pathway for disposing of misfolded proteins and damaged organelles. We demonstrated that SQSTM1 was cleaved following CVB3 infection through the proteolytic activity of viral proteinase 2Apro. The resulting cleavage fragments of SQSTM1 were no longer the substrates of autophagy, and their ability to form protein aggregates was greatly decreased due to incapability of interaction with ubiquitinated proteins. We further tested whether NBR1, a functional homolog of SQSTM1, can compensate for SQSTM1 loss-of-function after viral infection. Of interest, we found that NBR1 was also cleaved after coxsackievirus infection, excluding the possible compensation of NBR1 for the loss of SQSTM1. This cleavage took place at two sites mediated by virus-encoded proteinase 2Apro and 3Cpro, respectively. In addition to the loss-of-function, we showed that the C-terminal fragments of SQSTM1 and NBR1 exhibited a dominant-negative effect against native SQSTM1/NBR1, probably by competing for LC3 and ubiquitin chain binding. Apart from the disruption of selective autophagy, CVB3 infection also impaired autophagic flux as confirmed by flux assays with a combination of a tandem fluorescence-tagged LC3 stable cell line and a non-cleavable construct of SQSTM1. Finally, we studied the roles of SQSTM1 and NBR1 in autophagic degradation of depolarized mitochondria (referred to as mitophagy). Following mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler to trigger mitophagy, we demonstrated that NBR1 did not appear to be required for mitochondrial clustering. Deficiency of NBR1 alone or in concert with SQSTM1 did not block the clearance of damaged mitochondria, suggesting that NBR1 is dispensable for mitophagy regardless of the status of SQSTM1. Taken together, the findings in this study suggest novel mechanisms in coxsackieviral pathogenesis: coxsackievirus infection induces abnormal accumulation of ubiquitin conjugates through the disruption of selective degradation of protein aggregates and blockage of autophagic flux.

View record

Viral myocarditis, the inflammation of myocardium initiated by viral infection, is an important cause of mortality in neonates and children. In addition, it is a precursor to dilated cardiomyopathy (DCM). To date, no effective therapy is available for viral myocarditis/DCM. Coxsackievirus B3 (CVB3) is an important human pathogen of viral myocarditis. Extensive research efforts on CVB3 have broadened our understanding of the virus-host protein interactions. However, the pathogenesis of coxsackievirus-induced myocarditis is not fully understood. The objective of this dissertation is to explore the role of host protein manipulation in coxsackieviral replication and pathogenicity. My hypotheses are that (1) coxsackievirus hijacks host’s cellular autophagy mechanism to facilitate its own replication; and (2) the serum response factor (SRF) is cleaved by viral protease 2A during coxsackievirus infection and contributes to impaired myocardial function and progression to DCM. For project 1, I demonstrated that CVB3 manipulates the host autophagy pathway to supplement viral replication. Autophagy is an evolutionary conserved homeostatic mechanism in eukaryotes that degrades and recycles long-lived cytoplasmic proteins, as well as damaged organelles. The hallmark of autophagy is the formation of double-membrane vesicles known as autophagosomes. I provided the initial evidence that CVB3 infection induces the formation of autophagosomes. Up-regulation of autophagosome formation enhances CVB3 replication, whereas downregulation of autophagy pathway reduces CVB3 replication. My results help clarify the nature of the intracellular membranes previously shown to be required for viral replication. For project 2, I demonstrated that CVB3 manipulates SRF expression via protein cleavage. SRF is a transcription factor vital for the expression of cardiac contractile/regulator genes, as well as gene silencing microRNAs. Cardiac-specific knockout of SRF in adult transgenic mice results in disruption of cardiac gene expression and development of severe DCM. I showed that SRF is cleaved in CVB3-infected mouse hearts and cardiomyocytes. Further studies revealed that SRF is cleaved at the 327 amino acids position by CVB3-encoded protease 2A. I demonstrated that SRF cleavage contributes to DCM by abolishing the transactivation property of SRF and generating dominant-negative SRF-truncates. Taken together, these novel viral strategies bridged existing knowledge and may serve as therapeutic targets for viral myocarditis/DCM.

View record

Viral myocarditis, an inflammatory disease of the myocardium, can lead to the development of dilated cardiomyopathy (DCM), a common cause of heart failure. Coxsackievirus B3 (CVB3) in the family of Picornaviridae is one of the primary causative agents of viral myocarditis. The ubiquitin/proteasome system (UPS), a primary intracellular protein degradation system in eukaryotic cells, has emerged as a key modulator in viral infectivity and virus-mediated pathogenesis. Our laboratory has previously demonstrated a potential role of the UPS in CVB3 infection. However, the effect of proteasome inhibition on CVB3-induced myocarditis in vivo has not been assessed and the underlying mechanism by which the UPS regulates CVB3 replication remains unclear. In this dissertation, my hypothesis is that the UPS plays a critical role in the pathogenesis of CVB3-induced myocarditis through promoting CVB3 replication and by regulating host protein degradation. To test this hypothesis, I proposed three aims. In aim 1, using a myocarditis-susceptible mouse model, I demonstrated that treatment with a proteasome inhibitor MLN353 significantly attenuates CVB3-induced myocardial damage, suggesting that proteasome inhibition may provide a therapeutic means for viral myocarditis. During this study, however, the potential toxicity of general inhibition of proteasome was recognized, which prompted me to search for the specific targets within the UPS utilized by CVB3. In aim 2, collaborating with others, I showed that protein ubiquitination is enhanced and CVB3 protein 3D is ubiquitinated during viral infection. Gene-silencing of ubiquitin significantly reduces viral titers. However, this reduction is not as potent as by proteasome inhibition, suggesting that ubiquitin-independent proteasomal degradation may also play a role during CVB3 infection.In aim 3, I showed that REG gamma, which mediates ubiquitin-independent protein degradation, enhances CVB3 replication via facilitating p53 degradation. During CVB3 infection, REG gamma is sumoylated and translocated.Taken together, the results suggest a therapeutic value of proteasome inhibition in the treatment of viral myocarditis. The data also demonstrate important roles of both the ubiquitin-dependent and -independent pathways in the regulation of CVB3 infection. Identification of the specific substrates within the UPS during CVB3 infection and the potential mechanisms involved allows for more precise targeting in drug therapy.

View record