Structural insight into the unique properties of adeno-associated virus serotype 9

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.     

Identification of the galactose binding domain of the adeno-associated virus serotype 9 capsid

Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Matrix metalloproteinase 9 facilitates West Nile virus entry into the brain

West Nile virus (WNV) is the most-common cause of mosquito-borne encephalitis in the United States. Invasion of the brain by WNV is influenced by viral and host factors, and the molecular mechanism underlying disruption of the blood-brain barrier is likely multifactorial. Here we show that matrix metalloproteinase 9 (MMP9) is involved in WNV entry into the brain by enhancing blood-brain barrier permeability. Murine MMP9 expression was induced in the circulation shortly after WNV infection, and the protein levels remained high even when viremia subsided. In the murine brain, MMP9 expression and its enzymatic activity were upregulated and MMP9 was shown to partly localize to the blood vessels. Interestingly, we also found that cerebrospinal fluid from patients suffering from WNV contained increased MMP9 levels. The peripheral viremia and expression of host cytokines were not altered in MMP9(-/-) mice; however, these animals were protected from lethal WNV challenge. The resistance of MMP9(-/-) mice to WNV infection correlated with an intact blood-brain barrier since immunoglobulin G, Evans blue leakage into brain, and type IV collagen degradation were markedly reduced in the MMP9(-/-) mice compared with their levels in controls. Consistent with this, the brain viral loads, selected inflammatory cytokines, and leukocyte infiltrates were significantly reduced in the MMP9(-/-) mice compared to their levels in wild-type mice. These data suggest that MMP9 plays a role in mediating WNV entry into the central nervous system and that strategies to interrupt this process may influence the course of West Nile encephalitis.     

Glycan binding avidity determines the systemic fate of adeno-associated virus type 9

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析.结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%.RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定.利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp protease proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性.     

狂犬病病毒SRV_9突变株的构建与拯救

为构建人工修饰的狂犬病病毒,首先用人细胞色素C基因替换狂犬病病毒SRV9株基因间隔区中的非必需区域Ψ区并缺失基因组全长cDNA的糖蛋白CD编码区,得到突变型SRV9cDNA质粒。然后,该质粒与表达野生型SRV9四种结构蛋白N、P、G和L的质粒共转染BHK-21细胞。免疫荧光试验显示转染细胞中有大量特异性荧光,电子显微镜观察可见大量典型的狂犬病病毒粒子。上述结果表明已成功地拯救出了人工修饰的狂犬病病毒。狂犬病病毒SRV9突变株的成功构建与拯救,为新型狂犬病减毒活疫苗的研究提供了重要的实验工具。     

Pseudorabies virus Us9 directs axonal sorting of viral capsids

Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.     

狂犬病病毒SRV9疫苗株糖蛋白核酸序列及抗原特性研究

狂犬病病毒SRV9株是经克隆获得的中等蚀斑口服弱毒疫苗候选株,具有安全和免疫原性较好等。本试验根据狂犬病病毒糖蛋白核苷酸5'末端和3'末端序列设计了一对引物,通过反转录－聚合酶链式反应（RT－PCR）分别扩增了SRV9及其母源株SAD B19糖蛋白的全长cDNA,测序结果表明,SRV9和SAD B19糖蛋白cDNA读框长1575bp,编码524氨基酸残基的多肽。通过和其母源株SAD B19核苷酸序列比较发现,SRV9析158,575,931位碱基分别出现了G→A,A→G和A→G的转换,相应地导致第53位,192位,311位氨基酸出现了Gly→Glu,His→Arg,Thr→Ala突变。和我国现行的和疫苗株ERA的核苷酸和氨基酸比较,有10个碱其不同,7个氨基酸发生改变。经Jameson-Wolf抗原表位优势图分析发现。SRV9在192位氨基酸的改变,导致该部位产生了一个新的潜在抗原位点。由于狂犬病病毒糖蛋白是诱导机体产生中和抗体唯一抗原,因此,上述氨基酸的改变可能与其特殊的蚀斑特性和其安全性提高有关。保持对毒株的氨基酸序列分析也是监测其特性的重要手段之一。     

狂犬病病毒SRV9疫苗株糖蛋白核酸序列及抗原特性研究

狂犬病病毒SRV9株是经克隆获得的中等蚀斑口服弱毒疫苗候选株,具有安全和免疫原性较好等优点.本试验根据狂犬病病毒糖蛋白核苷酸5'末端和3'末端序列设计了一对引物,通过反转录-聚合酶链式反应(RT-PCR)分别扩增了SRV9及其母源株SAD B19糖蛋白的全长cDNA.测序结果表明,SRV9和SAD B19糖蛋白cDNA读框长1575bp,编码524氨基酸残基的多肽.通过和其母源株SAD B19核苷酸序列比较发现,SRV9的158、575、931位碱基分别出现了G→A、A→G和A→G的转换,相应地导致第53位、192位、311位氨基酸出现了Gly→Glu、His→Arg、Thr→Ala突变;和我国现行的犬用疫苗株ERA的核苷酸和氨基酸比较,有10个碱基不同,7个氨基酸发生改变.经Jameson-Wolf抗原表位优势图分析发现,SRV9在192位氨基酸的改变,导致该部位产生了一个新的潜在抗原位点.由于狂犬病病毒糖蛋白是诱导机体产生中和抗体唯一抗原,因此,上述氨基酸的改变可能与其特殊的蚀斑特性和其安全性提高有关.保持对毒株的氨基酸序列分析也是监测其特性的重要手段之一.     

The structure and function of the outer coat protein VP9 of Banna virus

Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 A resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked beta sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.     

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.     

Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAV(UK) viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.     

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.