The Myxoma Virus M-T5 Ankyrin Repeat Host Range Protein Is a Novel Adaptor That Coordinately Links the Cellular Signaling Pathways Mediated by Akt and Skp1 in Virus-Infected Cells

Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.Myxoma virus (MYXV) is a rabbit-specific poxvirus that has proven to be a useful model system to study the mechanism by which virus-encoded immunoregulatory proteins function to manipulate the various host immune responses during the course of viral infection (50). In its long-term evolutionary host (Sylvilagus sp.), MYXV causes a benign disease localized to the site of inoculation, but when the virus infects European rabbits (Oryctolagus cuniculus), it causes a rapid systemic and highly lethal infection called myxomatosis (13). The success of MYXV as a pathogen can be attributed to the ability of the virus to effectively avoid recognition and clearance by the immune systems of susceptible rabbit hosts. At the level of individual virus-infected cells, poxviruses, like MYXV, are particularly adept at binding and entering most mammalian cells, where they attempt to establish a favorable intracellular environment, which promotes viral replication. Thus, the ability of poxviruses to reconfigure or disable the various host antiviral responses of the infected cell directly dictates the outcome of a viral infection at the cellular level (28). To this end, poxviruses possess a large genomic capacity, and all encode a unique repertoire of immunoregulatory and host-interactive proteins that have evolved to specifically mediate a broad range of cellular processes critical for successful viral replication. To date, a large collection of poxvirus-encoded immunoregulatory proteins have been identified and characterized, including virokines, viroreceptors, signaling modulators, and inhibitors of various antiviral responses, such as apoptotic pathways and interferon signaling (43). More recently, a novel category of poxvirus ankyrin (ANK) repeat proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection (48, 49, 53).With the exception of poxviruses, the ANK motif is not commonly reported in viruses, although numerous examples have been identified in eukaryotic, bacterial, and archaeal proteins (6). The ANK motif, a tandemly repeated consensus module of approximately 33 amino acid residues, has been demonstrated to mediate diverse protein-protein interactions between cellular proteins having a broad spectrum of functional roles (32, 42). Solved crystal structures have revealed a conserved fold structure of the ANK repeat unit, by which each repeat forms a characteristic helix-loop-helix structure with a beta-hairpin/loop region projecting out from the helices at a 90° angle (3, 16, 19, 26). However, the ANK fold appears to be defined by its structure rather than any conserved biological function since there is no specific conserved substrate or binding partner structure that is universally recognized by members of the superfamily.The majority of poxviral ANK repeat-containing proteins also include a conserved carboxy-terminal PRANC (pox protein repeats of ankyrin C terminus) motif, which closely resembles a cellular protein motif called the F-box domain (30). Characterized as substrate adaptors, F-box-containing host proteins function to recruit cellular substrate proteins to the SCF ubiquitin-ligase complex (named after their main components, Skp1, cullin 1 [CUL1], and an F-box protein), where the substrates selected by the complex are ubiquitinated and targeted for degradation by the proteasome (21, 45, 60). The process of selective ubiquitination is an essential regulatory step for many cellular processes, and the human genome encodes more than 70 different F-box proteins, which collectively are thought to specifically target a broad collection of cellular substrates for delivery to the SCF complex to initiate turnover (62).Accounting for the largest family of poxviral proteins, almost all chordopoxviruses encode multiple ANK repeat-containing proteins, some of which have been defined as viral host range or virulence factors (30). For example, canarypox virus encodes 51 ANK repeat proteins, accounting for greater than 20% of the genome; however, most other poxviruses express less than a half dozen ANK repeat proteins (52). MYXV encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which have been described as virulence factors for myxomatosis in rabbits (5, 8, 33). The MYXV host range factor M-T5 was first characterized for its ability to regulate viral tropism within rabbit lymphocytes and, later, some classes of human cancer cell lines (33, 51). In human cancer cells, the direct physical interaction between M-T5 and the host cell Akt was shown to be a key restriction determinant for MYXV tropism in a subset referred to as type II cancer cells (56). Furthermore, M-T5 was shown to be functionally interchangeable with a host ANK repeat protein called PIKE-A, and the activation of Akt by either the viral M-T5 or the host PIKE-A protein was critical for MYXV permissiveness in type II human cancer cells (57). M-T5 was also demonstrated to protect MYXV-infected cells from virus-induced cell cycle arrest, a property which was linked to its ability to interact with a member of the host cell SCF complex called CUL1 (20). Unlike M-T5, no specific host binding partners or target substrates have yet been identified for M148, M149, or M150. However, in tumor necrosis factor alpha (TNF-α)-stimulated cells, M150 was shown to colocalize in the nucleus with NF-κB p65, suggesting that this MYXV protein may modulate the NF-κB pathway (8).In this study, we demonstrate that M-T5, M148, M149, and M150 all have functional carboxy-terminal PRANC/F-box-like domains and that each one can interact directly with the Skp1 component of the host SCF complex. We further examined the various binding domains of M-T5 and identified ANK repeats I and II as being important for interaction with Akt, whereas the PRANC/F-box-like domain was essential for binding to Skp1. We also show that the previously reported interaction of M-T5 with CUL1 was in fact, indirect linking of M-T5 to the host SCF complex via Skp1. More specifically, we investigated the ability of M-T5 to function as a molecular scaffold to link disparate cellular binding partners together within a single complex and report that the viral protein binds Akt and the SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we demonstrate that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during MYXV infection. These results suggest that ANK/PRANC proteins, such as M-T5, play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for viral replication.     

Crystal Structure of Vaccinia Viral A27 Protein Reveals a Novel Structure Critical for Its Function and Complex Formation with A26 Protein

Vaccinia virus envelope protein A27 has multiple functions and is conserved in the Orthopoxvirus genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21–84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner in vitro in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly in vitro. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.     

Functional Interaction of Human Immunodeficiency Virus Type 1 Vpu and Gag with a Novel Member of the Tetratricopeptide Repeat Protein Family

Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.     

Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI₁C or NI₃C) between an N-terminal capping repeat and mutants of the C-cap. An NI₁C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI₃C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.     

Insertion of Vaccinia Virus C7L Host Range Gene into NYVAC-B Genome Potentiates Immune Responses against HIV-1 Antigens

Background

The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice.

Methodology/Principal Findings

In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B.

Conclusions/Significance

These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.     

2019新型冠状病毒S蛋白的结构和功能分析

运用生物信息学,预测急性呼吸系统综合症冠状病毒2(SARS-CoV-2/2019-nCoV)的基本理化性质、结构、功能和抗原表位等,为新型冠状病毒肺炎(COVID-19)的防治提供思路。应用ExPASy分析S蛋白的消光系数、不稳定系数和半衰期等理化性质;利用SignaIP v5.0分析S蛋白的信号肽;应用TMHMM分析S蛋白的跨膜区;利用NetPhos3.1在线工具预测S蛋白的磷酸化位点;应用Pfam预测S蛋白的结构域;应用PSIPRED分析S蛋白的二级结构特征;利用SWISS-MODEL构建S蛋白的三级结构;利用BLAST分析SARS-CoV-2的S蛋白与其他物种的相似性;利用MEGA软件分析2019-nCoV的S蛋白与其他物种的进化关系。S蛋白由1 273个氨基酸组成,其相对分子质量为141 178.47,等电点为6.24,含有一个跨膜区,是低亲水性分泌蛋白;S蛋白的基本组成单位为纤突蛋白,其二级结构中以无规则卷曲和螺旋结构为主,三级结构中纤突糖蛋白和ACE2复合体具有重要的意义;2019-nCoV与蝙蝠冠状病毒和SARS-CoV同源;S蛋白存在多个潜在的线性T细胞和B细胞表位,1 202～1 210位氨基酸区域的抗原性和应答频率最高。生物信息学技术有利于了解S蛋白的理化性质、结构、功能和潜在的线性T细胞表位等,可为新型冠状肺炎的研究和防治提供参考依据。     

微丝相关新蛋白hHBRK1相互作用蛋白质的鉴定

为了鉴定hHBRK1的相互作用蛋白,通过DNA重组构建重组表达质粒pGEXhHBRK1,并以谷胱甘肽Sepharose4B亲合层析法,获得纯化的重组融合蛋白GSThHBRK1.以小鼠心肌组织为研究对象,采用GSTpulldown技术结合Western印迹法,证实hHBRK1与小鼠心肌肌钙蛋白TEa亚型(EacTnT)相互作用.结果提示,hHBRK1与EacTnT结合,可能参与心肌微丝的聚合,为小鼠cTnT众多的剪接体,提供了一种可能的功能定位.     

牛泡沫病毒调节蛋白功能及其在LTR上应答元件的研究

牛泡沫病毒（ＢＦＶ）具有复杂的基因组结构,其基因组除编码反转录病毒共有的ｇａｇ、ｐｏｌ、ｅｎｖ三个结构基因之外,在其ｅｎｖ和３’ＬＴＲ之间有两个重叠的读码框（ＯＲＦ－１和ＯＲＦ－２）,编码Ｂｏｒｆ－１、Ｂｏｒｆ－２、Ｂｅｔ等多种调节蛋白,其中Ｂｏｒｆ－１（２４９ａａ）为ＢＦＶ反式激活因子（Ｔａｓ）。为研究Ｂｏｒｆ－１的结构与功能,Ｂｏｒｆ－１在ＬＴＲ上的应答元件及作用机制,Ｂｏｒｆ－２、Ｂｅｔ等调     

Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-Å resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.     

Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.     

Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.     

A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 3₁₀ helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.     

Defective Vaccinia Virus as a Biologically Safe Tool for the Overproduction of Recombinant Human Secretory Proteins

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.