Xiaoyu Li

The MOV10 Helicase Inhibits LINE-1 Mobility

Background: Retrotransposon LINE-1 causes dozens of genetic diseases. Results: Human MOV10 diminishes the level of LINE-1 RNA by acting at a post-transcriptional stage. Conclusion: The host protein suppresses LINE-1 transposition. Significance: MOV10 contributes to the cellular control of LINE-1 replication. LINE-1 (long interspersed element 1) is an autonomous non-long terminal repeat retrotransposon. Its replication often causes mutation and rearrangement of host genomic DNA. Accordingly, host cells have evolved mechanisms to control LINE-1 mobility. Here, we report that a helicase named MOV10 effectively suppresses LINE-1 transposition. Mutating the helicase motifs impairs this function of MOV10, suggesting that MOV10 requires its helicase activity to suppress LINE-1 replication. Further studies show that MOV10 post-transcriptionally diminishes the level of LINE-1 RNA. The association of MOV10 with both LINE-1 RNA and ORF1 suggests that MOV10 interacts with LINE-1 RNP and consequently causes its RNA degradation. These data demonstrate collectively that MOV10 contributes to the cellular control of LINE-1 replication.

Characterization of the interface of the bone marrow stromal cell antigen 2-Vpu protein complex via computational chemistry.

Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of enveloped viruses from the cell surface. Various viral counter measures have been discovered, which allow viruses to escape BST-2 restriction. Human immunodeficiency virus type 1 (HIV-1) encodes viral protein U (Vpu) that interacts with BST-2 through their transmembrane domains and causes the downregulation of cell surface BST-2. In this study, we used a computer modeling method to establish a molecular model to investigate the binding interface of the transmembrane domains of BST-2 and Vpu. The model predicts that the interface is composed of Vpu residues I6, A10, A14, A18, V25, and W22 and BST-2 residues L23, I26, V30, I34, V35, L41, I42, and T45. Introduction of mutations that have been previously reported to disrupt the Vpu-BST-2 interaction led to a calculated higher binding free energy (MMGBSA), which supports our molecular model. A pharmacophore was also generated on the basis of this model. Our results provide a precise model that predicts the detailed interaction occurring between the transmembrane domains of Vpu and BST-2 and should facilitate the design of anti-HIV agents that are able to disrupt this interaction.

Structure-based design of novel chemical modification of the 3'-overhang for optimization of short interfering RNA performance.

Short interfering RNAs (siRNAs) are broadly used to manipulate gene expression in mammalian cells. Although chemical modification is useful for increasing the potency of siRNAs in vivo, rational optimization of siRNA performance through chemical modification is still a challenge. In this work, we designed and synthesized a set of siRNAs containing modified two-nucleotide 3-overhangs with the aim of strengthening the interaction between the 3-end of the siRNA strand and the PAZ domain of Ago2. Their efficiency of binding to the PAZ domain was calculated using a computer modeling program, followed by measurement of RNA-Ago2 interaction in a surface plasmon resonance biochemical assay. The results suggest that increasing the level of binding of the 3-end of the guiding strand with the PAZ domain, and/or reducing the level of binding of the sense strand through modifying the two-nucleotide 3-overhangs, affects preferential strand selection and improves siRNA activity, while we cannot exclude the possibility that the modifications at the 3-end of the sense strand may also affect the recognition of the 5-end of the guiding strand by the MID domain. Taken together, our work presents a strategy for optimizing siRNA performance through asymmetric chemical modification of 3-overhangs and also helps to develop the computer modeling method for rational siRNA design.

The cellular source for APOBEC3G's incorporation into HIV-1

Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear. Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation. Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.

A novel peptide to disrupt the interaction of BST‐2 and Vpu

Bone marrow stromal cell antigen 2 (BST‐2) inhibits the release of HIV‐1 and other enveloped viruses from the cell surface. HIV‐1 Vpu binds to BST‐2 through an interaction between transmembrane domains (TMD) of the two proteins and induces the downregulation of cell surface BST‐2, thereby counteracting its antiviral activity. In this study, we designed and prepared a modified peptide BST2‐TM‐P1, which include the sequence of BST‐2 TMD, keeping its property competing with BST‐2 to bind with Vpu. Biological assay results indicate BST2‐TM‐P1 could increase the BST‐2 level at the cell surface in Vpu dependent manner and significantly inhibit the replication of HIV‐1 virion. Our studies indicate that blocking the interaction of Vpu and BST‐2 is an effective way to combat HIV‐1 infection.

A small molecule compound IMB-LA inhibits HIV-1 infection by preventing viral Vpu from antagonizing the host restriction factor BST-2.

Human BST-2 inhibits HIV-1 replication by tethering nascent virions to the cell surface. HIV-1 codes Vpu that counteracts BST-2 by down-regulating this restriction factor from the cell surface. This important function makes Vpu a potential therapeutic target. Yet, no agents have been reported to block Vpu from antagonizing BST-2. In this study, we report a small molecule compound IMB-LA that abrogates the function of Vpu and thereby strongly suppresses HIV-1 replication by sensitizing the virus to BST-2 restriction. Further studies revealed that IMB-LA specifically inhibits Vpu-mediated degradation of BST-2 and restores the expression of BST-2 at the cell surface. Although IMB-LA does not prevent Vpu from interacting with BST-2 or β-TrCP2-containing ubiquitin E3 ligase, sorting of BST-2 into lysosomes in Vpu-expressing cells is blocked by IMB-LA. Most importantly, HIV-1 release and infection is inhibited by IMB-LA only in BST-2-expressing cells. In summary, results herein demonstrated that IMB-LA could specifically inhibit the degradation of BST-2 induced by Vpu, and impair HIV-1 replication in a BST-2 dependent manner, suggesting the feasibility of utilizing small molecule compounds to disable the antagonist function of Vpu and thereby expose HIV-1 to the restriction by BST-2.

A cell-based high-throughput approach to identify inhibitors of influenza A virus.

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus. Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors. In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA, which expresses Gaussia luciferase upon influenza A virus infection. Using this cell line, an assay was developed and optimized to search for inhibitors of influenza virus replication. Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format (Z′ factor value>0.8). A pilot screening provides further evidence for validation of the assay. Taken together, this work provides a simple, convenient, and reliable HTS assay to identify compounds with anti-influenza activity.

Identification and characterization of loop7 motif and its role in regulating biological function of human APOBEC3G through molecular modeling and biological assay

Human APOBEC3G (hA3G) is a cytidine deaminase which inhibits HIV-1 replication. The HIV-1 accessory protein viral infectivity factor (Vif) counteracts with hA3G by targeting it for proteasomal degradation. In this work, we constructed and optimized molecular models of the hA3G dimer and the hA3G–Vif complex. The molecular modeling study revealed that the loop7 motif of hA3G appears on the interfaces of both the hA3G–Vif complex and the hA3G dimer. Biochemical analysis provided evidence suggesting that binding of Vif to hA3G results in steric blocking of hA3G dimerization, implying that monomeric hA3G serves as a substrate for Vif-mediated degradation. Furthermore, we presented evidence for the important roles of the loop7 motif, especially the central residues within the region, in hA3G dimerization, hA3G--Vif interaction, Vif-mediated hA3G degradation as well as subcellular localization of hA3G. This work highlights a multiple-task interface formed by loop7 motif, which regulates biological function of hA3G, thus providing the feasibility of the strategy of blocking Vif-mediated A3G degradation by targeting the putative site around loop7.

Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase

Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg2+∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation.

AbstractBackgroudnBST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface.ResultsUsing a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and β-TrCP2, but decreases BST-2 ubiquitination.ConclusionOur study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.