Analysis of Subcellular Localization and Pathogenicity of Plum Bark Necrosis Stem-Pitting Associated Virus Protein P6

Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.

Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS: The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantitative PCR was used to measure the expression of IL-2, IFN-gamma, IL-4 and IL-10 in the allografts. RESULTS: The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-gamma, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62 +/- 0.09,1.52 +/- 0.11,1.50 +/- 0.07 and 1.43 +/- 0.07 versus 1.29 +/- 0.09, 1.32 +/- 0.07, 1.34 +/- 0.06 and 1.35 +/- 0.04, respectively). CONCLUSIONS: pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological findings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

Targeting adeno-associated virus and adenoviral genetherapy for hepatocellular carcinoma

Human hepatocellular carcinoma(HCC)heavily endangers human heath worldwide.HCC is one of most frequent cancers in China because patients with liver disease,such as chronic hepatitis,have the highest cancer susceptibility.Traditional therapeutic approaches have limited efficacy in advanced liver cancer,and novel strategies are urgently needed to improve the limited treatment options for HCC.This review summarizes the basic knowledge,current advances,and future challenges and prospects of adeno-associated virus(AAV)and adenoviruses as vectors for gene therapy of HCC.This paper also reviews the clinical trials of gene therapy using adenovirus vectors,immunotherapy,toxicity and immunological barriers for AAV and adenoviruses,and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors.

Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA

Cyclin dependent kinases （cdks） play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus （rAAV） encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA （rAAV-EGFP-U6-cdc2-siRNA） was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous sys- tem. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plas- mids were encapsidated as recombinant virions. PCl2 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus or AAV vectors by South- ern blot and transcript situation or foreign genes by dot blot. Results The hybridization tests showed that AAV in- troduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PCl2 cells. Conclusion AAV vectors can serve as high efficiency vectors or target genes in neuronal PC12 cells.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Salvianolic acid B increases recombinant adeno-associated virus transduction through redirecting trafficking pathways and stabilizing perinuclear accumulations

OBJECTIVE To screen small molecule compounds from traditional Chinese medicine that can enhance recombinant adeno-associated virus(rAAV) transduction.METHODS Recombinant adeno-associated virus(rAAV) has been established as a powerful tool for in vivo gene transfer and achieved much promise in gene therapy applications.However,widespread clinical use has been limited by transduction efficiency.In the current study,we screened a panel of small molecule compound from traditional Chinese medicine focused on AAV intracellular trafficking process and found salvianolic acid B can significantly enhance rAAV2 transduction.RESULTS Salvianolic acid B caused a dose-depen.dent increase in rAAV2 transduction regardless of vector dose,genome architecture,and over a broad range of cell line from various cell type and species(HEK293,HeLa,HepG2,Huh-7,CHO-K1,LO-2).Salvianolic acid B treatment redirected rAAV2 particles toward large vesicles positive for late endosomal(Rab7) and lysosomal(LAMP1) markers.Furthermore,salvianolic acid B acted to increase accumulation of viral particles at the perinuclear region.CONCLUSION In summary,our results suggest that salvi.anolic acid B redirects rAAV2 toward more productive trafficking pathways and stabilizes perinuclear accumulations of vectors,facilitating productive nuclear trafficking.

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION： The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.

Adeno-associated virus vectors for human gene therapy

Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.

Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus （rAAAV）, AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein （GFP） gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast （CEF） or chicken embryonic liver （CEL） cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek＇s disease virus （MDV）, avian adenovirus, chicken embryo lethal orphan （CELO） virus or infectious bursal disease virus （IBDV）. Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein （gfp） expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV- mediated transgene expression could be enhanced by super infection with the helper viruses.

Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

A study on the integration of adeno-associated virus inverted terminal repeat fold structure in eukaryotic chromosome in vitro

Adeno-associated virus(AAV) is a single-stranded linear defective parvovirus. Using the model proposed by Cavalier-Smith for replication of ss-DNA virus. the linear fragment of inverted terminal repeats(ITRs) from PAAV/neo was isolated, denatured and refolded over. The daughter DNA was synthesized by using 3'-OH terminus as a primer with the new strand covalently linked to the parental strand. After the palindromic structure with neomycin resistance gene controlled by SV40 early promoter into eukaryotic cells. Southern blot showed that the molecule of hairpin structure could facilitate the copies into mammalian cells in vitro. The experiment supports the Cavalier-Smith's replication model of ss-DNA virus and provides another new system for DNA transduction.

Effects of recombinant adeno-associated viruses expressing human vascular endothelial growth factor 165 on spinal cord injury

Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses （rAAV）-hVEGF165-IRES-human recombinant green fluorescent protein （hrGFP） （AAV-VEGF） and rAAV-IRES-hrGFP （AAV-GFP）. Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.

Recombinant adeno-associated virus delivered human thioredoxin-PR39 prevents hypoxia-induced apoptosis of ECV304 cells

Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.

Construction of recombinant adeno-associated virus vector co-expressing hVEGF_(165) and hBMP_7 gene

Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recombination. Methods：The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results：Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion：Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.

CONSTRUCTION AND EXPRESSION OF ADENO-ASSOCIATED VIRUS-BASED PLASMID EXPRESSING VECTORS CONTAINING hIL-2 GENE OR mIFN-γ GENE

Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.

single nucleotide polymorphism in theEpstein-Barr virus genome is strongly associatedwith a high risk of nasopharyngeal carcinoma

Background Epstein-Barr virus （EBV） commonly infects the general population and has been associated withnasopharyngeal carcinoma （NPC）, which has a high incidence in certain regions.This study aimed to address how EBVvariations contribute to the risk of NPC.Methods： Using logistic regression analysis and based on the sequence variations at EBV-encoded RPMSh a multistageassociation study was conducted to identify EBV variations associated with NPC risk. A protein degradationassay was performed to characterize the functional relevance of the RPMS1 variations.Results： Based on EBV-encoded RPMS1 variations, a single nucleotide polymorphism （SNP） in the EBV genome（locus 155391： G〉A, named G155391 A） was associated with NPC in 157 cases and 319 healthy controls from an NPCendemic region in South China [P 〈 0.001, odds ratio （OR） = 4.47,95% confidence interval （Cl） 2.71-7.37].The resultswere further validated in three independent cohorts from the NPC endemic region （P 〈 0.001, OR = 5.20,95% Cl3.18-8.50 in 168 cases vs. 241 controls, and P 〈 0.001, OR = 5.27,95% Cl 4.06-6.85 in 726 cases vs. 880 controls） anda non-endemic region （P 〈 0.001, OR = 7.52,95% Cl 3.69-1532 in 58 cases vs. 612 controls）.The combined analysisin 1109 cases and 2052 controls revealed that the SNP G155391A was strongly associated with NPC （Pcomb/ned 〈 0.001,OR = 5.27,95% Cl 4.31-6.44）. Moreover, the frequency of the SNP G155391A was associated with NPC incidence butwas not associated with the incidences of other EBV-related malignancies. Furthermore, the protein degradation assayshowed that this SNP decreased the degradation of the oncogenic RPMS1 protein.Conclusions： Our study identified an EBV variation specifically and significantly associated with a high risk of NPCThese findings provide insights into the pathogenesis of NPC and strategies for prevention.