Browsing by Author "Fan, Yanlin"

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    Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA–DNA pairing
    (Oxford University Press, 2016) Lin, Jiangguo; Countryman, Preston; Chen, Haijiang; Pan, Hai; Fan, Yanlin; Jiang, Yunyun; Kaur, Parminder; Miao, Wang; Gurgel, Gisele; You, Changjiang; Piehler, Jacob; Kad, Neil M.; Riehn, Robert; Opresko, Patricia L.; Smith, Susan; Tao, Yizhi Jane; Wang, Hong
    Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.
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    Orsay Virus CP-δ Adopts a Novel β-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber
    (American Society for Microbiology, 2020) Guo, Yusong R.; Fan, Yanlin; Zhou, Ying; Jin, Miao; Zhang, Jim L.; Jiang, Hongbing; Holt, Matthew V.; Wang, Tao; Young, Nicolas L.; Wang, David; Zhong, Weiwei; Tao, Yizhi Jane
    Fiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematode Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ(1–101) (a deletion mutant containing the first 101 amino acid [aa] residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple β-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. The δ proteins from two other nematode viruses, Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.
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    Orsay δ protein is required for non-lytic viral egress
    (American Society for Microbiology, 2018) Yuan, Wang; Zhou, Ying; Fan, Yanlin; Tao, Yizhi Jane; Zhong, Weiwei
    Nonenveloped gastrointestinal viruses, such as human rotavirus, can exit infected cells from the apical surface without cell lysis. The mechanism of such nonlytic exit is poorly understood. The nonenveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans Dye staining results suggested that Orsay virus exits from the intestine of infected worms in a nonlytic manner. Therefore, the Orsay virus-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay virus genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein, δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had a normal CP-δ fusion but could not produce free δ due to the lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and colocalized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay virus infection rearranged ACT-5 apical localization. Reduction of the ACT-5 level via RNA interference (RNAi) significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay virus exit. Together, these data support a model in which the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate the polarized, nonlytic egress of Orsay virus.IMPORTANCE: An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain nonenveloped viruses can exit cultured cells in nonlytic ways; however, such nonlytic exit has not been demonstrated in vivo In addition, it is not clear how such nonlytic exit is achieved mechanistically in vivo Orsay virus is a nonenveloped RNA virus that infects the intestine cells of the nematode C. elegans It is currently the only virus known to naturally infect C. elegans Using this in vivo model, we show that the δ protein encoded by Orsay virus facilitates the nonlytic exit of the virus, possibly by interacting with host actin on the apical side of worm intestine cells.
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    Structure and Function Studies of the Orsay Virus δ Protein
    (2018-10-31) Fan, Yanlin; Tao, Yizhi Jane
    Despite the wide use of Caenorhabditis elegans (C. elegans) as a model organism, the first virus naturally infecting this organism was not discovered until seven years ago. The Orsay virus has a bipartite, positive sense RNA genome, with the RNA 1 segment encoding the RNA-dependent RNA polymerase (RdRP) and the RNA 2 segment encoding the capsid protein (CP) and another protein named delta (δ). δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but their structure and function were mostly unknown. To get a better understanding of the infection mechanism of this novel virus, I performed both structural and functional studies of the free δ protein as well as the CP-δ fusion protein. Using a combination of techniques including electron microscopy, X-ray crystallography, computational and biophysical analyses, we found that the Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal coiled coil, a β-stranded filament in the middle, and a C-terminal head domain. The N-terminal coiled coil of the polypeptide is essential for the proper folding and oligomerization of the pentameric structure. Interestingly, δ from the other two nematode viruses, namely Le Blanc and Santeuil viruses, also forms pentameric fibrous molecules similar to the Orsay δ. The relatively low amino acid sequence identities of ~30-40% among these δ proteins likely account for several notable structural differences among these fibers. For example, the internal globular domain observed in Orsay is not clearly visible in both Le Blanc and Santeuil fibers. Also, Santeuil fiber tends to bend in the middle, which does not apply to the other two δ fibers. On the other hand, we found that free δ is required for non-lytic egress of Orsay virus from worm intestinal cells. Transgenic worms over-expressing free δ showed that δ was preferably localized to the apical membrane of host intestine cells, where it likely facilitates virus release by reconstructing the terminal web network. The co-localization of δ and worm ACT-5 and their interaction suggest that Orsay δ may functionally interact with ACT-5 to mediate the viral exit from the worm intestine. For the CP-δ protein, both recombinant Orsay capsid containing CP-δ and purified Orsay virions are associated with protruding long fibers with globular heads at the distal end, suggesting that CP-δ protein can be incorporated into the Orsay capsid during assembly. To test the biological function of the capsid-associated CP-δ fibers, mutant viruses with disrupted fiber structures were generated by organism-based reverse genetics. These viruses were found to be either non-viable or poorly infectious according to phenotypic and qRT-PCR analyses. In addition, by performing a protein competition assay, we found that adding purified full-length δ protein into the culture medium could inhibit Orsay infection, while adding an N-terminal fragment δ(1-101) had no such effect. Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ may interact with the host cellular receptors via its C-terminal head to mediate Orsay entry into worm intestine cells. Therefore, my study suggested that the Orsay δ protein likely has two independent functions at different stages of the virus life cycle. Considering the similarity between the worm and human intestinal cell morphologies, it is expected that my findings will lead to a better understanding of the infection mechanism of not only Orsay, but also gastrointestinal viruses infecting both mammals and humans.