Shaoyan Zhang

All Publications

• IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease JOURNAL OF HEPATOLOGY Park, J., Zhao, Y., Zhang, F., Zhang, S., Kwong, A. C., Zhang, Y., Hoffmann, H., Bushweller, L., Wu, X., Ashbrook, A. W., Stefanovic, B., Chen, S., Branch, A. D., Mason, C. E., Jung, J. U., Rice, C. M., Wu, X. 2023; 78 (1): 45-56

  Abstract

  A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD.To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development.Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity.Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD.An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

  View details for DOI 10.1016/j.jhep.2022.08.022

  View details for Web of Science ID 000923633900006

  View details for PubMedID 36049612

  View details for PubMedCentralID PMC9772150

• Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs JOURNAL OF VIROLOGY Zhang, S., Sun, R., Carvalho, C., Han, J., Zheng, L., Qu, F. 2021; 95 (18): e0016921

  Abstract

  Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.

  View details for DOI 10.1128/JVI.00169-21

  View details for Web of Science ID 000708638400003

  View details for PubMedID 34160262

  View details for PubMedCentralID PMC8387050

• Bottleneck, Isolate, Amplify, Select (BIAS) as a mechanistic framework for intracellular population dynamics of positive-sense RNA viruses VIRUS EVOLUTION Qu, F., Zheng, L., Zhang, S., Sun, R., Slot, J., Miyashita, S. 2020; 6 (2): veaa086

  Abstract

  Many positive-sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolates viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution and inspire new solutions to virus disease mitigation.

  View details for DOI 10.1093/ve/veaa086

  View details for Web of Science ID 000610104700043

  View details for PubMedID 33343926

  View details for PubMedCentralID PMC7733609

• Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus VIRUSES-BASEL Sun, R., Zhang, S., Zheng, L., Qu, F. 2020; 12 (3)

  Abstract

  RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.

  View details for DOI 10.3390/v12030350

  View details for Web of Science ID 000525486800012

  View details for PubMedID 32235750

  View details for PubMedCentralID PMC7150753

• Superinfection Exclusion by p28 of Turnip Crinkle Virus Is Separable from Its Replication Function MOLECULAR PLANT-MICROBE INTERACTIONS Guo, Q., Zhang, S., Sun, R., Yao, X., Zhang, X., Tatineni, S., Meulia, T., Qu, F. 2020; 33 (2): 364-375

  Abstract

  We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28-p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.

  View details for DOI 10.1094/MPMI-09-19-0258-R

  View details for Web of Science ID 000511839400025

  View details for PubMedID 31880982

• Repression of turnip crinkle virus replication by its replication protein p88 VIROLOGY Zhang, S., Sun, R., Guo, Q., Zhang, X., Qu, F. 2019; 526: 165-172

  Abstract

  We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild-type p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance.

  View details for DOI 10.1016/j.virol.2018.10.024

  View details for Web of Science ID 000452934300019

  View details for PubMedID 30391806

• A New Mechanistic Model for Viral Cross Protection and Superinfection Exclusion FRONTIERS IN PLANT SCIENCE Zhang, X., Zhang, S., Guo, Q., Sun, R., Wei, T., Qu, F. 2018; 9: 40

  Abstract

  Plants pre-infected with a mild variant of a virus frequently become protected against more severe variants of the same virus through the cross protection phenomenon first discovered in 1929. Despite its widespread use in managing important plant virus diseases, the mechanism of cross protection remains poorly understood. Recent investigations in our labs, by analyzing the whole-plant dynamics of a turnip crinkle virus (TCV) population, coupled with cell biological interrogation of individual TCV variants, revealed possible novel mechanisms for cross protection and the closely related process of superinfection exclusion (SIE). Our new mechanistic model postulates that, for RNA viruses like TCV, SIE manifests a viral function that denies progeny viruses the chance of re-replicating their genomes in the cells of their "parents," and it collaterally targets highly homologous superinfecting viruses that are indistinguishable from progeny viruses. We further propose that SIE may be evolutionarily selected to maintain an optimal error frequency in progeny genomes. Although primarily based on observations made with TCV, this new model could be broadly applicable to other viruses as it provides a molecular basis for maintaining virus genome fidelity in the face of the error-prone nature of virus replication process.

  View details for DOI 10.3389/fpls.2018.00040

  View details for Web of Science ID 000423350400001

  View details for PubMedID 29422912

  View details for PubMedCentralID PMC5788904

• A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus PLOS PATHOGENS Zhang, X., Sun, R., Guo, Q., Zhang, S., Meulia, T., Halfmann, R., Li, D., Qu, F. 2017; 13 (3): e1006253

  Abstract

  Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.

  View details for DOI 10.1371/journal.ppat.1006253

  View details for Web of Science ID 000398120300035

  View details for PubMedID 28267773

  View details for PubMedCentralID PMC5357057