Entire Glycoprotein Research Articles

Discordant humoral and T cell immune responses to SARS-CoV-2 vaccination in people with multiple sclerosis on anti-CD20 therapy.

BackgroundSphingosine-1-phosphate receptor (S1P) modulators and anti-CD20 therapies impair humoral responses to SARS-CoV-2 mRNA vaccines. Relatively few studies have assessed the impact of an array of disease modifying therapies (DMTs) for multiple sclerosis (MS) on T cell immune responses to SARS-CoV-2 vaccination.MethodsIn 101 people with MS, we measured humoral responses via an immunoassay to measure IgG against the COVID-19 spike S1 glycoprotein in serum. We also measured T cell responses using FluoroSpot assay for interferon gamma (IFN-γ) (Mabtech, Sweden) using cryopreserved rested PBMCs and then incubated in cRPMI with 1µg/ml of pooled peptides spanning the entire spike glycoprotein (Genscript, 2 pools; 158 peptides each). Plates were read on an AID iSpot Spectrum to determine the number of spot forming cells (SFC)/106 PBMCs. We tested for differences in immune responses across DMTs using linear models.FindingsHumoral responses were detected in 22/39 (56.4%) participants on anti-CD20 and in 59/63 (93.6%) participants on no or other DMTs. In a subset (n=88; 87%), T cell responses were detected in 76/88 (86%), including 32/33 (96.9%) participants on anti-CD20 therapies. Anti-CD20 therapies were associated with an increase in IFN-γ SFC counts relative to those on no DMT or other DMTs (for anti-CD20 vs. no DMT: 425.9% higher [95%CI: 109.6%, 1206.6%] higher; p<0.001; for anti-CD20 vs. other DMTs: 289.6% [95%CI: 85.9%, 716.6%] higher; p<0.001).InterpretationWe identified a robust T cell response in individuals on anti-CD20 therapies despite a reduced humoral response to SARS-CoV-2 vaccination. Follow up studies are needed to determine if this translates to protection against COVID-19 infection.FundingThis study was funded partially by 1K01MH121582-01 from NIH/NIMH and TA-1805-31136 from the National MS Society (NMSS) to KCF and TA-1503-03465 and JF-2007-37655 from the NMSS to PB. This study was also supported through the generosity of the collective community of donors to the Johns Hopkins University School of Medicine for COVID research.

Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe acute human disease with lethal outcome. The knowledge about the immune response for this human health threat is highly limited. In this study, we have screened the glycoprotein of CCHFV for novel linear B-cell epitopic regions using a microarray approach. The peptide library consisted of 168 synthesized 20mer peptides with 10 amino acid overlap covering the entire glycoprotein. Using both pooled and individual human sera from survivors of CCHF disease in Turkey five peptide epitopes situated in the mucin-like region and GP 38 (G15-515) and GN G516-1037 region of the glycoprotein were identified as epitopes for a CCHF immune response. An epitope walk of the five peptides revealed a peptide sequence located in the GN region with high specificity and sensitivity. This peptide sequence, and a sequence downstream, reacted also against sera from survivors of CCHF disease in South Africa. The cross reactivity of these peptides with samples from a geographically distinct region where genetically diverse strains of the virus circulate, enabled the identification of unique peptide epitopes from the CCHF glycoprotein that could have application in development of diagnostic tools. In this study clinical samples from geographically distinct regions were used to identify conserved linear epitopic regions of the glycoprotein of CCHF.

Deletion of a Predicted β-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons.

We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted β-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

The Role of Sarcospan in Cardiac Sarcolemma Organization and Function

Mutations in the dystrophin gene cause loss of dystrophin and the entire glycoprotein complex (DGC) that protects muscle cells from contraction-induced damage. Therapeutic interventions for Duchenne muscular dystrophy (DMD) have targeted small protein components of the DGC, like sarcospan (SSPN), to stabilize the membrane complex. Previously, we showed that increased SSPN expression in a DMD mouse model (mdx3.0) attenuates skeletal muscle pathology in DMD by increasing DGC interactions and cellular-adhesion. However, the effects of SSPN overexpression on cardiomyopathy in the mdx mouse remains unknown. SSPN overexpression in WT (WT3.0) and mdx background enhanced levels of DGC at the cardiac sarcolemma and attenuated DMD cardiac tissue pathology. Furthermore, anatomical and molecular studies showed that overexpression of SSPN reduced EBD uptake, CK-MB, cardiac myopathy markers such as atrial natriuretic peptide (ANP) and reduced left ventricular (LV) mass. These findings support the concept that alterations in sarcospan expression contributes to cardiac dysfunction and re-introduction of sarcospan could be used as a molecular target to prevent or attenuate cardiomyopathy associated with DMD. NRSA GM07104 NRSA T32AR059033 CureDuchenne Fellowship William Townsend Porter Pre-doctoral Fellowship from the American Physiological Society NIH/NIAMS- R01 AR048179 and UL1TR000124

Bioinformatics Study on Zaire Ebolavirus (EBOV) Protein for Better Understanding the Vaccine Development

Nine, Ebola viruse EBOV (Zaire ebolavirus), proteins are extracted from the NCBI repository and their study was carried out. The physicchemical properties and evolutionary link with other such viruses by homology modeling were carried out. All the proteins show rich in leucine domain an ideal requirement for fast attachment of the virus to the receptor molecule on the host cell surface. The prediction of trans-membrane sequence for the entire glycoprotein component reveals the ability of the virus to enter the host with ease. The lack of adequate homology model for the viral proteins indicates its novel origin and lack of well traceable evolutionary link. We studied the homology based model by using various available tools and find similar approach in all, hence finally concentrated only on one method. The model predicted shows well acceptable region on Ramchandran plot. This discrepancy is only due to the fact that we validate the model to Ramchandran plot and the model predicted were not under the well acceptable ‘e’ value range i.e. &gt;1. Therefore we suggests that vaccine production against this deadly virus should be concentrated on the structure and functions of glycoprotein like low quality secreted glycoprotein (NP_066248), low quality spike glycoprotein(NP_066246)0, small secreted glycoprotein (NP_066247) and RNA dependent RNA polymerase (NP_066251). DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11588Int J Appl Sci Biotechnol, Vol. 2(4): 544-552

아시아에서 분리된 viral haemorrhagic septicaemia virus (VHSV) isolates의 계통분석학적 비교

바이러스성 출혈성 패혈증을 일으키는 VHSV는 국내의 넙치양식에 심각한 피해를 주고 있다. 본 연구에서는 우리나라에서 분리한 8종의 VHSV 분리주들의 glycoprotein (G)의 염기서열을 분석하여 Korean VHSV 분리주들과 기보고된 일본 및 중국의 분리주들을 계통발생학적으로 비교하여 G-protein의 8 부위(G34, C528, A755, T834, T951, T1147, T1221, T1336)에서 국내 VHSV 분리주들의 특이적인 서열을 확인하였고, 전 세계적으로 분리된 VHSV의 분리주들과의 비교결과 8 부위 중 3 부위 (A755, T834, T1221)의 국내VHSV 분리주 특이적 염기서열들을 확인하였다. 또한 중국 유래 VHSV 분리주의 특이적 염기서열 11 부위 (C498, C545, C548, C642, G648, T678, A738, C804, C834, G851, C1139)와 일본 유래 VHSV 분리주의 특이적 염기서열 1부위 (C179)를 확인하였다. 이상의 연구결과는 국내 특이적인 염기서열을 확보하여, 구역화된 수산생물 질병의 예찰, 모니터링 및 질병 관리에 유용하게 사용될 것으로 사료된다. Viral haemorrhagic septicaemia virus (VHSV), the causative agent of viral haemorrhagic septicaemia (VHS), is an epidemic virus of cultured olive flounder Paralichthys olivaceus in Korea. In the present study, the entire glycoprotein (G) gene including several hypervariable regions from 36 isolates of diverse geographic and host origin and 8 Korean VHSV isolates from cultured olive flounder were analyzed. Phylogenetic analysis indicated that most of Asian VHSV belong to the genotype IVa group, suggesting that they originated from a common ancestral virus. Comparative sequence analysis of the complete G protein from Korean VHSV isolates revealed 3 Korean strain-specific nucleotide residues (nucleotide number of G-region: A755, T834 and T1221). These results suggest that Korean VHSV originated from a common ancestor, but these regional specific nucleotide sequences suggest that genetic differences of VHSV are more related to geographic areas than to host fish species.

Characterization of perch rhabdovirus (PRV) in farmed grayling Thymallus thymallus

Two Finnish fish farms experienced elevated mortality rates in farmed grayling Thymallus thymallus fry during the summer months, most typically in July. The mortalities occurred during several years and were connected with a few neurological disorders and peritonitis. Virological investigation detected an infection with an unknown rhabdovirus. Based on the entire glycoprotein (G) and partial RNA polymerase (L) gene sequences, the virus was classified as a perch rhabdovirus (PRV). Pairwise comparisons of the G and L gene regions of grayling isolates revealed that all isolates were very closely related, with 99 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis demonstrated that they were closely related to the strain isolated from perch Perca fluviatilis and sea trout Salmo trutta trutta caught from the Baltic Sea. The entire G gene sequences revealed that all Finnish grayling isolates, and both the perch and sea trout isolates, were most closely related to a PRV isolated in France in 2004. According to the partial L gene sequences, all of the Finnish grayling isolates were most closely related to the Danish isolate DK5533 from pike. The genetic analysis of entire G gene and partial L gene sequences showed that the Finnish brown trout isolate ka907_87 shared only approximately 67 and 78% identity, respectively, with our grayling isolates. The grayling isolates were also analysed by an immunofluorescence antibody test. This is the first report of a PRV causing disease in grayling in Finland.

Structural glycobiology of the major allergen of Artemisia vulgaris pollen, Art v 1: O-glycosylation influence on the protein dynamics and allergenicity

Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen. It is formed by an N-terminal globular defensin-like part and a C-terminal proline-rich domain. As the structure and the dynamics of Art v 1 have been mostly described for its recombinant, non-glycosylated form, which does not occur in normal plant physiology, the present work intends to obtain a three-dimensional model for Art v 1 native O-glycosylation structure and to evaluate the influence of such glycans over the protein dynamics and allergenicity through molecular dynamics simulations in triplicates. Structural insights into the mutual recognition of Art v 1 protein and carbohydrate moieties recognition by antibodies were obtained, in which glycan chains remained close to the previously identified epitopes in the defensin-like domain, thus pointing to potential interferences with antibodies recognition. To our knowledge, this is the first structural report of an entire furanose-containing glycoprotein. As well, together with the previously determined NMR structures, the obtained results contribute in the comprehension of the effect of glycosylation over both proline-rich and defensin-like domains, providing an atomic representation of such alterations.

An Assembly Model of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a bunyavirus endemic to Africa and the Arabian Peninsula that infects humans and livestock. The virus encodes two glycoproteins, Gn and Gc, which represent the major structural antigens and are responsible for host cell receptor binding and fusion. Both glycoproteins are organized on the virus surface as cylindrical hollow spikes that cluster into distinct capsomers with the overall assembly exhibiting an icosahedral symmetry. Currently, no experimental three-dimensional structure for any entire bunyavirus glycoprotein is available. Using fold recognition, we generated molecular models for both RVFV glycoproteins and found significant structural matches between the RVFV Gn protein and the influenza virus hemagglutinin protein and a separate match between RVFV Gc protein and Sindbis virus envelope protein E1. Using these models, the potential interaction and arrangement of both glycoproteins in the RVFV particle was analyzed, by modeling their placement within the cryo-electron microscopy density map of RVFV. We identified four possible arrangements of the glycoproteins in the virion envelope. Each assembly model proposes that the ectodomain of Gn forms the majority of the protruding capsomer and that Gc is involved in formation of the capsomer base. Furthermore, Gc is suggested to facilitate intercapsomer connections. The proposed arrangement of the two glycoproteins on the RVFV surface is similar to that described for the alphavirus E1-E2 proteins. Our models will provide guidance to better understand the assembly process of phleboviruses and such structural studies can also contribute to the design of targeted antivirals.

GROMOS96 43a1 performance in predicting oligosaccharide conformational ensembles within glycoproteins

In previous work [Pol-Fachin, L.; Fernandes, C. L.; Verli, H.; Carbohydr. Res. 2009, 344, 491–500], we had demonstrated that GROMOS96 43a1 force field and Löwdin HF/6-31G∗∗-derived atomic charges, adequately represent a glycoprotein’s conformational ensemble in aqueous solutions, taking as the starting geometries NMR-determined structures. Based on such data, the present work intends to evaluate the use of the main solution conformations of isolated disaccharides, to build the carbohydrate moiety of glycoproteins, for which no previous experimental information is available. The observed results suggested that the entire glycoprotein scaffold appears unable to promote major modifications in the conformational behavior of glycosidic linkages. Additionally, when compared to energy contour plots, the results support the use of solution ensembles, to refine vacuum conformations of carbohydrate databases in the assembling of glycoproteins 3D structures. Finally, such approach is applied to build a full glycosylated model for COX-1 and COX-2 enzymes.

Genetic diversity of Chinese rabies viruses: Evidence for the presence of two distinct clades in China

There have been three major rabies epidemics in China since the 1950s. To gain more insights into the molecular epidemiology of rabies viruses (RVs) for the third (the current) epidemic, we isolated RV from dogs and humans in major endemic areas, and characterized these isolates genetically by sequencing the entire glycoprotein (G) gene and the G–L non-coding region. These sequences were also compared phylogenetically with RVs isolated in China during previous epidemics and those around the world. Comparison of the entire G genes among the Chinese isolates revealed up to 21.8% divergence at the nucleotide level and 17.8% at the amino acid level. The available Chinese isolates could be divided into two distinct clades, each of which could be further divided into six lineages. Viruses in clade I include most of the Chinese viruses as well as viruses from southeast Asian countries including Indonesia, Malaysia, the Philippines, Thailand, and Vietnam. The viruses in the other clade were found infrequently in China, but are closely related to viruses distributed worldwide among terrestrial animals. Interestingly, most of the viruses isolated during the past 10 years belong to lineage A viruses within clade I whereas most of the viruses isolated before 1996 belong to other lineages within clades I and II. Our results indicated that lineages A viruses have been predominant during the past 10 years and thus are largely responsible for the third and the current epidemic in China. Our results also suggested that the Chinese RV isolates in clade I share a common recent ancestor with those circulating in southeast Asia.

Characteristics of HIV Type 1 (HIV‐1) Glycoprotein 120envSequences in Mother‐Infant Pairs Infected with HIV‐1 Subtype CRF01_AE

We analyzed the characteristics of the envelope genes of human immunodeficiency virus type 1 in 17 mother-infant pairs infected with variants of the CRF01_AE clade. A total of 353 sequences covering almost the entire glycoprotein (gp) 120 region were available for analysis. We found that, even if the virus population in the mother was complex, only viruses of a restricted subset were transmitted to her infant, independently of whether transmission occurred in utero or during the intrapartum period. We did not find that shorter gp120 regions or fewer potential N-glycosylation sites (PNGS) were characteristic of viruses transmitted from mother to infant. However, our data suggest that a limited number of PNGS that seem to be conserved in all variants in infants but are not uniformly present in variants in mothers may confer an advantage for transmission of the virus, thereby highlighting the potentially important role of the "glycan shield." This finding was particularly significant for the PNGS at positions N301 and N384.

Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene

Twelve Brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (IBV) were propagated in embryonating chicken eggs. The entire S1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RT-PCR-RFLP), using the restriction enzymes HaeIII, XcmI and BstyI. The RFLP patterns led to the classification of these isolates into five distinct genotypes: A, B, C, D and Massachusetts. Five of twelve isolates were grouped in Massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: A (2), B (2), C (2) or D (1). Such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the IBV strains that are circulating in Brazilian commercial poultry flocks.

Isolation of viral haemorrhagic septicaemia virus from muskellunge, Esox masquinongy (Mitchill), in Lake St Clair, Michigan, USA reveals a new sublineage of the North American genotype

Viral haemorrhagic septicaemia virus (VHSV) was isolated from muskellunge, Esox masquinongy (Mitchill), caught from the NW portion of Lake St Clair, Michigan, USA in 2003. Affected fish exhibited congestion of internal organs; the inner wall of the swim bladder was thickened and contained numerous budding, fluid-filled vesicles. A virus was isolated using fish cell lines inoculated with a homogenate of kidney and spleen tissues from affected fish. Focal areas of cell rounding and granulation appeared as early as 24 h post-inoculation and expanded rapidly to destroy the entire cell sheet by 96 h. Electron microscopy revealed virions that were 170-180 nm in length by 60-70 nm in width having a bullet-shaped morphology typical of rhabdoviruses. The virus was confirmed as VHSV by reverse transcriptase-polymerase chain reaction. Sequence analysis of the entire nucleoprotein and glycoprotein genes revealed the virus was a member of the North American genotype of VHSV; however, the isolate was sufficiently distinct to be considered a separate sublineage, suggesting its origin may have been from marine species inhabiting the eastern coastal areas of the USA or Canada.

The N-Terminal SRCR-SID Domain of gp-340 Interacts with HIV Type 1 gp120 Sequences and Inhibits Viral Infection

Proteins encoded by the SRCR superfamily including gp340 recognize repeated patterns on pathogenic microorganisms and play important roles in innate immune defense as well as epithelial cell differentiation. Based upon the presence of SRCR domains in proteins with broad binding specificities and high amino acid sequence homology, it was speculated that SRCR domains may be involved in ligand binding. In this study, a truncated gp340 molecule representing the N-terminal sequence including the first SRCR and one-half of the first SID was expressed in mammalian 293 cells as a 35-kDa recombinant protein. The expressed protein was recognized by a panel of antibodies specific for human salivary agglutinin (SAG) and the full-length parental gp340 and exhibited biological properties similar to the entire 340-kDa glycoprotein. The truncated gp340 protein bound to the same HIV-1 V3 sequences previously identified to interact with full-length SAG in a Ca2+ -dependent manner. The recombinant N-terminal SRCR protein also demonstrated potent anti-HIV- 1 activity against both CCR5- and CXCR4-using isolates, similar to the full-length glycoprotein. We have, thus, demonstrated that the N-terminal SRCR of gp340 directly interacts with viral gp120 and likely mediates anti-HIV-1 activity via this interaction.

Glycoproteomics: protein modifications for versatile functions

This satellite meeting of the Thirtieth FEBS Congress and the Ninth IUBMB Conference was held in Dubrovnik, Croatia, between 28 and 30 June 2005. The conference was organized by G. Lauc, J. Dumic and M. Flogel from the University of Zagreb, Croatia. ![][1] Glycosylation is perhaps the most common and versatile post‐translational modification of proteins. It is estimated that well over one‐half of all mammalian proteins are glycosylated (Apweiler et al , 1999). The roles of protein glycans, whether N ‐ or O ‐linked forms, are varied. Glycan is required for the biological function of certain proteins, such as the Fc‐effector function of immunoglobulin G (IgG), the regulated clearance of the glycohormone lutropin (LH), the targeting of lysosomal enzymes and controlling the circulation lifetime of glycoprotein drugs (for example, erythropoietin). Glycans also serve as recognition targets for their complementary binding proteins, the lectins. This family includes self proteins that are involved in such diverse functions as fertilization, development, differentiation and lymphocyte circulation, as well as in the development of pathological conditions. Glycans are also used as sites of attachment by microbes and toxins, and often constitute the first contact point between pathogen and host. In addition, glycans serve subtle functions, such as protecting proteins from proteolytic enzymes, promoting protein folding into proper tertiary and quaternary structures, and modulating the biological activities of proteins. Other forms of glycans, such as glycolipids and proteoglycans, also have important structural roles, as well as acting as recognition targets. Glycomics is the investigation and documentation of the large collection of glycan structures. The function of a glycan can vary depending on the protein to which it is attached and the precise site of attachment. It is imperative, therefore, to document the structure and function of the entire glycoprotein. This integrated approach of compiling glycoprotein structure and function … [1]: /embed/graphic-1.gif

793. Efficient Expression of the 6 kb Mini-Dystrophin Gene by Trans-Splicing Adeno-Associated Viral (AAV) Vector Restores the Entire Dystrophin-Associated Glycoprotein Complex and Reduces Contraction-Induced Damage in the Mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common lethal muscle disease caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) has been used to express the 3.8 kb C-terminal deleted micro-dystrophin gene for DMD gene therapy. Despite amelioration of some disease-associated changes, the microgene is not fully functional. Trans-splicing AAV vectors have been developed recently to express the 6 kb H2-R19 mini-dystrophin gene, the most competent gene besides the full-length gene. Our recent studies suggest that the RNA processing represents a critical barrier in trans-splicing AAV vector. In this study, we first screened a series of endogenous exon/intron/exon junctions by the RNase protection assay (RPA). The 56/56/57, 60/60/61 and 63/63/64 junctions hold the highest theoretic splicing values and may represent the most favorable sites to split the dystrophin gene. We generated synthetic mini-constructs carrying the splicing signals from each of above junctions. We also inserted the inverted terminal repeat junction in the middle of the intron. As a control, we included the 53/53/54 junction that has a low splicing value. The most efficient splicing was achieved in the 63/63/64 junction where the ratio of the spliced to unspliced RNA (S/U ratio) reached 7.1 0.9. The 60/60/61 and the 53/53/54 junctions yielded medium S/U ratios of 3.0 0.4 and 2.5 0.3, respectively. The 56/56/57 junction resulted in the lowest S/U ratio of 1.0 0.6. We also quantified the level of accumulated mRNA. Surprisingly, the highest mRNA was obtained from the 60/60/61 junction (255.1 20.6, relative unit), followed by the 63/63/64 (139.5 6.4), the 53/53/54 (48.8 20.6) and the 56/56/57 (0.8 0.2) junctions. We next generated two sets of AAV-6 trans-splicing vectors based on the 60/60/61 and the 63/63/64 junctions, respectively. 2 1010 particles of each set of vectors were delivered to the anterior tibialis (TA) muscle of 2-m-old mdx mice. Mini-dystrophin expression was quantified at 1 m post-infection by imunofluorescence staining. Consistent with the RPA results, trans-splicing vectors based on the 60/60/61 junction yielded 34 fold higher expression. To further evaluate the therapeutic potential, we delivered the 60/60/61 trans-splicing vectors (donor/acceptor co-infection, or donor or acceptor single infection) to both the TA and the extensor digitorium longus (EDL) muscles of the 2-m-old mdx mice. At 3 m post-infection, transduction efficiency in co-infected TA muscle reached 60-80%. Importantly, the entire dystrophin-associated glycoprotein complex (including dystroglycan, sarcoglycan, dystrobrevin and syntrophin) was restored in co-infected muscle. Furthermore, trans-splicing vector mediated mini-dystrophin expression provided significant protection against eccentric contraction induced injury in the EDL muscle.

The domains of glycoprotein D required to block apoptosis depend on whether glycoprotein D is present in the virions carrying herpes simplex virus 1 genome lacking the gene encoding the glycoprotein.

An earlier report showed that viruses lacking the open reading frames encoding glycoproteins J and D but containing the glycoprotein D in their envelopes (gD-/+ stocks) and viruses lacking both the open reading frames and the glycoproteins in their envelopes (gD-/- stocks) induce apoptosis (G. Zhou, V. Galvan, G. Campadelli-Fiume, and B. Roizman, J. Virol. 74:11782-11791, 2000). Furthermore, apoptosis was blocked by delivery in trans of genes expressing glycoprotein D or J. Whereas gD-/- stocks attach but cannot initiate productive infection, gD-/+ stocks infect cells and produce gD-/- progeny virus. The difference in the infectivity of these two stocks suggested the possibility that the requirements for blocking apoptosis may be different. To test this hypothesis, we cloned into baculoviruses the entire wild-type glycoprotein D (Bac-gD-WT), the ectodomain only (Bac-gD-A), the ectodomain and the transmembrane domain (Bac-gD-B), the ectodomain and the cytoplasmic domain without the transmembrane domain (Bac-gD-C), or the transmembrane domain and the carboxyl-terminal cytoplasmic domain (Bac-gD-D). We report the following. Apoptosis induced by gD-/+ stocks was blocked by delivery in trans of recombinant baculovirus Bac-gD-WT, Bac-gD-A, Bac-gD-B, or Bac-gD-C but not of Bac-gD. Apoptosis induced by gD-/- stocks was blocked by Bac-gD-WT or by a mixture of Bac-gD-B and Bac-gD-D but not by any baculoviruses expressing truncated glycoprotein D alone or by the mixture of Bac-gD-A and Bac-gD-D. We conclude that the requirements to block apoptosis induced by the two virus stocks are different. The gD ectodomain is sufficient to block apoptosis induced by gD, whereas both the ectodomain and the cytoplasmic domain are required to block apoptosis induced by gD-/- stocks. The results indicate that in the case of gD-/- stocks, the transmembrane domain is required either to deliver the ectodomain to the appropriate intracellular compartment or to form multimeric constructs which virtually reconstitute gD through the interaction of transmembrane domains.

Spike Gene Analysis of the DE072 Strain of Infectious Bronchitis Virus: Origin and Evolution

The entire S2 gene of the DE072 strain of infectious bronchitis virus (IBV) was sequenced. The nucleotide and amino acid sequence was most similar to the D1466 strain and was 84.8% and 89.9% identity, respectively. The nucleotide and amino acid sequence similarity among the DE072 strain and other IBV strains was less than 71.9% and 76.6%, respectively. Phylogenetic analysis, based on both nucleotide and amino acid sequence, showed that IBV isolates were divided into two distinct groups. The DE072 strain clustered only with the D1466 strain, and all of the other strains were distinct from those two viruses. Further the nucleotide sequence analysis of the entire spike glycoprotein gene of the DE072 strain demonstrated that most of the gene contained a D1466-like sequence, and five putative cross-over sites were identified. Based on cross-over site, phylogenetic trees were constructed for different regions of the spike gene, and a difference in topology between these trees was observed. Considering the difference in S2 gene sequence identity and tree topology, we assume that DE072 and D1466 viruses share a different origin from other isolates of IBV. Furthermore, entire spike gene analysis indicates that the DE072 strain has undergone recombination event as well as extensive antigenic variation.