Jinming Zhou

The Interferon-Inducible MxB Protein Inhibits HIV-1 Infection

The interferon-inducible myxovirus resistance (Mx) proteins play important roles in combating a wide range of virus infections. MxA inhibits many RNA and DNA viruses, whereas the antiviral activity of MxB is less well established. We find that human MxB inhibits HIV-1 infection by reducing the level of integrated viral DNA. Passaging HIV-1 through MxB-expressing cells allowed the evolution of a mutant virus that escapes MxB restriction. HIV-1 escapes MxB restriction by mutating the alanine residue at position 88 in the viral capsid protein (CA), with a consequent loss of CA interaction with the host peptidylprolyl isomerase cyclophilin A (CypA), suggesting a role for CypA in MxB restriction. Consistent with this, MxB associates with CypA, and shRNA-mediated CypA depletion or cyclosporine A treatment resulted in the loss of MxB inhibition of HIV-1. Taken together, we conclude that human MxB protein inhibits HIV-1 DNA integration by a CypA-dependent mechanism.

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.

Identification of novel key amino acids at the interface of the transmembrane domains of human BST-2 and HIV-1 Vpu

BackgroundBST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits virus release by tethering viral particles to the cell surface. This antiviral activity of BST-2 is antagonized by HIV-1 accessory protein Vpu. Vpu physically interacts with BST-2 through their mutual transmembrane (TM) domains. In this study, we utilized the BRET assay and molecular dynamics (MD) simulation method to further characterize the interaction of BST-2 and Vpu.ResultsAmino acids I34, L37, P40 and L41 in the TM domain of BST-2, and L11, A18 and W22 in the TM domain of Vpu were identified to be critical for the interaction between BST-2 and Vpu. The residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu were shown, for the first time, to be important for their interaction. Furthermore, triple-amino-acid substitutions, 14–16 (AII to VAA) and 26–28 (IIE to AAA) in Vpu TM, not the single-residue mutation, profoundly disrupted BST-2/Vpu interaction. The results of MD simulation revealed significant conformational changes of the BST-2/Vpu complex as a result of mutating P40 of BST-2 and L11, 14–16 (AII to VAA) and 26–28 (IIE to AAA) of Vpu. In addition, disrupting the interaction between BST-2 and Vpu rendered BST-2 resistant to Vpu antagonization.ConclusionsThrough use of the BRET assay, we identified novel key residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu that are important for their interaction. These results add new insights into the molecular mechanism behind BST-2 antagonization by HIV-1 Vpu.

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.

Human APOBEC3F incorporation into human immunodeficiency virus type 1 particles

APOBEC3 proteins are a family of cytidine deaminases that exhibit broad antiretroviral activity. Among APOBEC3 proteins, APOBEC3G (hA3G) and APOBEC3F (hA3F) exhibit the most potent anti-HIV-1 activities. Although the incorporation of hA3F into virions is a prerequisite for exerting its antiviral function, the detail mechanism underlying remains incompletely understood. In this work, we present data showing that the nucleocapsid (NC) domain of HIV-1 Gag and a linker sequence between the two cytidine deaminase domains within hA3F, i.e., 104-156 amino acids, are required for viral packaging of hA3F. A detailed mapping study reveals that the cluster of basic residues surrounding the N-terminal zinc finger (ZF) and the linker region between the ZFs of HIV-1 NC play an important role in A3F incorporation, in addition, at least one of two ZFs is required. A hA3F fragment is able to compete with both hA3G and hA3F for viral incorporation, suggesting a common mechanism underlying virion encapsidation of hA3G and hA3F. Taken together, these results shed a light on the detail mechanism underlying viral incorporation of hA3F.

Characterization of and functional evidence for Ste27 of Streptomyces sp. 139 as a novel spermine/spermidine acetyltransferase

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has remarkable anti-rheumatoid arthritis activity in vivo and its biosynthesis gene cluster (ste) consists of 27 ORFs (open reading frames). The present paper reports our study of the protein product encoded by ste27. Database searching reveals the homology of Ste27 with some spermidine/spermine acetyltransferases. To confirm the prediction, the ste27 gene was cloned and expressed in Escherichia coli BL21(DE3) cells and recombinant Ste27 was purified. The following enzymatic analysis revealed its ability of transferring the acetyl group from acetyl-CoA to spermidine and spermine, with spermidine being the preferred substrate. Ste27 can acetylate the N1, N4 and N8 positions on spermidine. The Km values of Ste27 were determined for spermidine and spermine, as well as for acetyl-CoA, poly-L-lysine and glucosamine 6-phosphate. Upon gene knockout, the exopolysaccharide-27m produced by the mutant strain Streptomyces sp. 139 (ste27-), compared with Ebosin, exhibited a significantly reduced binding activity to the interleukin-1 receptor. After gene complementation, the binding activity was partially restored. This demonstrated that the ste27 gene is involved in the biosynthesis of Ebosin. Molecular modelling was also carried out to predict the binding mode of Ste27 with acetyl-CoA, spermidine or spermine.

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.

AbstractBackgroud BST-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.