【病毒外文文獻(xiàn)】2011 The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis co
《【病毒外文文獻(xiàn)】2011 The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis co》由會員分享,可在線閱讀,更多相關(guān)《【病毒外文文獻(xiàn)】2011 The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis co(7頁珍藏版)》請在裝配圖網(wǎng)上搜索。
RESEARCH Open Access The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus Christel Schwegmann We els 1 Sandra Bauer 1 Christine Winter 1 2 Luis Enjuanes 3 Hubert Laude 4 and Georg Herrler 1 Abstract Background Transmissible gastroenteritis virus TGEV has a sialic acid binding activity that is believed to be important for enteropathogenicity but that has so far appeared to be dispensable for infection of cultured cells The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions and comparison of TGEV strains and mutants as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity Methods The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5 20 and 60 min Prior to infection cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface or mock treated In a second approach pre treatment of the virus with porcine intestinal mucin was performed followed by the plaque assay after a 5 min adsorption time A student s t test was used to verify the significance of the results Results Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection However when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60 A TGEV PUR46 mutant HAD3 deficient in sialic acid binding showed a 77 lower titer than the parental virus after a 5 min adsorption time After an adsorption time of 60 min the titer of HAD3 was 58 lower than that of TGEV PUR46 Another TGEV strain TGEV Miller and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time Conclusions Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains Keywords coronavirus S protein sialic acid binding activity TGEV IBV cultured cells Background Enveloped viruses enter their target cells by a two step process 1 The initial event is the attachment of the virion to the cell surface Subsequently the viral envel ope fuses with the cellular membrane which enables the viral genome to get access to the cytoplasm The fusion reaction may occur at the plasma membrane or upon endocytotic uptake of the virion at the endosomal membrane The entry process requires the interaction of one or more viral surface proteins with cellular recep tors The binding to the cellular receptor mediates the attachment step and sets the stage for the subsequent fusion process Several viruses have developed a strategy to recognize more than one surface structure of the tar get cell The binding to some of these interaction part ners may not be sufficient for the virus to proceed to the fusion step but nevertheless it can support the entry Correspondence christel schwegmann tiho hannover de 1 Institute for Virology University of Veterinary Medicine Hannover B nteweg 17 30559 Hannover Germany Full list of author information is available at the end of the article Schwegmann We els et al Virology Journal 2011 8 435 2011 Schwegmann We els et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited process by making it more likely for the virus to find the actual cellular receptor TGEV is a porcine coronavirus that causes diarrhea in pigs of all ages 2 Piglets even die from the infection unless they are protected by maternal antibodies This enveloped virus with a positive stranded RNA genome enters cells using the glycoprotein S for both attachment to the cell surface and for fusion of the viral membrane with the cellular membrane The fusion activity of the S protein is induced only after interaction with a specific receptor on the surface of the target cell porcine ami nopeptidase N pAPN 3 The S protein is not only able to bind to pAPN it also has a sialic acid binding activity with a preference for N glycolylneuraminic acid 4 5 Interaction with sialylated macromolecules appears to be dispensable for infection of cultured cells but is believed to be important for the enteropathogenicity of the virus 6 7 This is based on the finding that a single mutation in the S protein may result in the loss of both the sialic acid binding activity and the enteropathogeni city whereas the mutant viruses can be propagated in cultured cells to the same titer as the parental virus 7 8 This finding has been explained by environmental conditions in the intestine that make it more difficult for a microorganism to initiate an intestinal infection compared to an infection of cultured cells 9 10 The intestinal epithelium is covered not only by a glycocalix layer but also by an even thicker layer of mucus 11 As mucins are rich in sialic acids they are interaction part ners for TGEV and thus may help to penetrate the mucus layer and to get access to pAPN on the surface of the intestinal epithelial cells 9 10 Results and discussion Comparison of infectivities with and without neuraminidase treatment We tried to obtain experimental evidence for a role of the sialic acid binding activity of TGEV in the infection of cells under unfavorable conditions For our analysis we used the Purdue strain of TGEV which was grown on swine testicular cells ST as described previously 5 We analyzed the effect of desialylation of cells on infec tion by TGEV by a plaque assay 8 In contrast to the regular plaque assay a neuraminidase treatment prior to infection was included to see a potential reduction in the number of plaques To evaluate the optimal experi mental setup different neuraminidase concentrations were included in the first analysis 0 50 125 250 500 1000 1500 mU ml data not shown A concentration of 250 mU ml resulted in a significant inhibition of TGEV infection at an adsorption time of 5 min As the cell cul ture appearance was not disturbed at this concentration we decided to use this neuraminidase concentration of 250 mU ml for subsequent experiments As higher concentrations of the enzyme up to 1 5 U ml gave no increase in virus inhibition we concluded that sialic acids were removed to a satisfactory level As shown in Figure 1 columns designated TGEV PUR46 and table 1 pre treatment of cells with neuraminidase from Clos tridium perfringens type V 250 mU ml Sigma for 60 min reduced the infectivity of the parental virus by 26 when the adsorption time was 60 min When the virus had only 5 min for adsorption the infectivity on desialy lated cells was reduced by 64 Infection at 20 min adsorption time was in between these two values reduc tion of 45 This result is consistent with a previous work where binding of virions rather than infectivity was analyzed and where we have shown that desialyla tion of cultured cells reduces the binding of TGEV par ticles to these cells 5 Our data indicate that the sialic acid binding activity increases the efficiency of infection at short adsorption times This conclusion is supported by the finding that no reduction was observed when the hemagglutination deficient mutant HAD3 was subjected to such an analysis Figure 1 table 1 This mutant has a point mutation in the S protein at position 209 Leu Pro and was selected because of its deficiency in sialic acid binding 8 Mutants m10 deletion of 4 amino acids at position 146 149 and m8 point mutation at amino acid 147 were analyzed in the same way for adsorption times of 5 and 60 min 6 7 In fact with these mutant viruses which are all deficient in sialic acid binding activity pre treatment of cells with neuramini dase even increased the infectivity This increase was significant for mutants HAD3 and m10 with a p value below 0 05 table 1 In a previous publication we con cluded that binding of the S protein to pAPN is even more efficient after neuraminidase treatment as sialic acid depletion on pAPN facilitates the protein binding 5 It is possible that this effect makes it is easier for the mutants to get access to the cellular receptor pAPN and to bind to the specific binding site after neuramini dase treatment of the cell culture In contrast in the porcine intestine the binding of the mutants to pAPN could be less efficient because of the presence of sialic acids For m8 and m10 it was reported that their entero pathogenicity is markedly reduced 6 7 Therefore a less efficient binding to the cellular receptor pAPN in vivo because of a deficiency in sialic acid binding could be one explanation for this reduced enteropathogenicity To include another TGEV strain in our study we ana lyzed the Miller strain 12 This strain was mainly pas saged in vivo has an enteric and respiratory tropism like the Purdue strain and is virulent in swine 12 13 As shown in Figure 2 and table 1 the Miller strain showed a reduction in infectivity after neuraminidase pre treatment of the cells This reduction is in the same range irrespective of the virion adsorption time 32 Schwegmann We els et al Virology Journal 2011 8 435 Page 2 of 7 reduction for 5 min 23 reduction for 20 min table 1 26 reduction for 60 min adsorption time Recently we have shown that infectious bronchitis virus IBV an avian coronavirus uses sialic acid as a receptor determinant for infection of both cultured cells and tracheal organ cultures 14 15 We were interested to know whether this virus also shows differences in the dependence on the sialic acid binding activity at short and long adsorption times As shown in Figure 2 and table 1 when analyzed in the same way as TGEV the Beaudette strain of IBV showed a reduction of the infec tivity after pre treatment of cells with neuraminidase Similar to the result obtained with the Miller strain the reduction was irrespective of a long or short adsorption time After 60 min adsorption the infectivity of IBV was reduced by 47 and after 5 min adsorption time it was reduced by 33 Taken together our results indicate that the Miller strain of TGEV rather resembles IBV than the Purdue strain of TGEV as far as the sialic acid dependence of infection is concerned Comparison of early and late infectivity The infectious titer at three different time points 5 20 60 min with and without neuraminidase treatment was calculated Figure 3 shows a mean value out of 4 differ ent experiments for TGEV PUR46 and HAD3 and out of 3 different experiments for TGEV Miller Highest titers 2 27 10 7 PFU ml at 5 min 3 71 10 7 PFU ml 0 50 100 150 200 250 56020 5 6060556020 TGEV PUR46 TGEV HAD3 TGEV m8 TGEV m10 infectivity time of adsorption min Figure 1 Sialic acid dependent infection by TGEV PUR46 The infectivity of parental virus left columns and the HAD3 mutant virus was determined for adsorption times of 5 20 or 60 min respectively The m8 and m10 mutant viruses were analyzed for adsorption times of 5 and 60 minutes Prior to infection ST cells were incubated for 60 min with either PBS grey columns or PBS containing 50 mU of neuraminidase white columns All experiments were performed 4 times with standard deviations shown at the corresponding columns Significant differences are marked with an asterisk p value 0 05 Table 1 Infectivities of virus strains and mutants after neuraminidase treatment of the cells Virus adsorption time 5 min 20 min 60 min TGEV PUR46 35 8 4 55 0 4 74 4 4 TGEV HAD3 149 1 4 144 1 4 130 7 4 TGEV m8 98 8 4 not determined 110 0 4 TGEV m10 119 9 4 not determined 109 3 4 TGEV Miller 67 7 3 76 9 3 74 0 3 IBV Beaudette 66 6 3 not determined 53 3 3 The significance of the value is indicated by an asterisk the number of independent experiments used for calculation is indicated in brackets 0 20 40 60 80 100 120 5560 60 IBV Beaudette TGEV Miller time of adsorption min infectivity 66 6 53 3 67 7 74 0 Figure 2 Sialic acid dependent infection by IBV Beaudette and TGEV Miller at short and long adsorption times The infectivity of IBV Beaudette and TGEV Miller was determined at adsorption times of 5 or 60 min respectively Prior to infection Vero cells for IBV and ST cells for TGEV were incubated for 60 min with either PBS grey columns or PBS containing 50 mU of neuraminidase white columns The experiments were performed 3 times Standard deviations are indicated Significant differences are marked with an asterisk p value 0 05 Schwegmann We els et al Virology Journal 2011 8 435 Page 3 of 7 at 20 min and 8 63 10 7 PFU ml at 60 min were obtained for TGEV PUR46 that increased over time After neuraminidase treatment the titers decreased sig nificantly for the first two time points 8 13 10 6 PFU ml at 5 min 2 06 10 7 PFU ml at 20 min and 7 33 10 7 PFU ml at 60 min Infectious titers for the hemag glutination deficient mutant HAD3 were 5 14 10 6 PFU ml at 5 min adsorption time 2 05 10 7 PFU ml at 20 min and 3 64 10 7 PFU ml at 60 min After neura minidase treatment titers increased 9 30 10 6 PFU ml at5min 2 96 10 7 PFU ml at 20 min and 4 74 10 7 PFU ml at 60 min For the TGEV Miller strain infec tious titers in cell culture were more than 10fold lower than for the TGEV Purdue strain 1 34 10 6 PFU ml at 5 min 2 09 10 6 PFU ml at 20 min 3 34 10 6 PFU ml at 60 min After neuraminidase treatment the titers of TGEV Miller decreased 9 08 10 5 PFU ml at 5 min 1 61 10 6 PFU ml at 20 min and 2 44 10 6 PFU ml at 60 min The differences in the titers between early 5 min and late 60 min infectivity of TGEV PUR46 HAD3 and TGEV Miller were statistically significant with p 0 05 The higher titers of TGEV PUR46 show that this virus strain is more cell culture adapted than the Miller strain The early infectivity 5 min adsorption time of HAD3 is about 22 6 of the value for TGEV PUR46 at this time point with p 0 014 However the late infectivity 60 min of HAD3 is about 42 2 of the value for TGEV PUR46 with p 0 021 This higher difference between TGEV PUR46 and HAD3 in early infectivity could be explained by the additional use of sialic acids for adsorption by TGEV PUR46 At short adsorption times the importance of sialic acid binding for infectivity is more pronounced than at longer adsorption times when binding to the cellular receptor pAPN compensated this phenomenon Reduction of TGEV infectivity by porcine intestinal mucins To find out if the sialic acid binding activity of the TGEV S protein can be inhibited by porcine intestinal mucins which are rich in sialic acids we performed a plaque assay after incubation of TGEV PUR46 with 5E 05 5E 06 5E 07 52060 1 2 3 4 5 6 1 TGEV PUR46 2 TGEV PUR46 NA 3 HAD3 NA 4 HAD3 5 TGEV Miller 6 TGEV Miller NA time of adsorption min infectivity PFU ml Figure 3 Comparison of early and late infectivity between TGEV PUR46 HAD3 and TGEV Miller The infectious titers of TGEV PUR46 HAD3 and TGEV Miller were calculated at adsorption times of 5 20 and 60 min respectively Infectivity was expressed in plaque forming units per ml PFU ml Prior to infection ST cells were incubated with either PBS 1 4 5 or PBS containing 50 mU of neuraminidase 2 3 6 100 100 8 77 1 37 6 0 20 40 60 80 100 120 infectivity 01 52 5 mucin concentration mg ml Figure 4 Mucin dependent infection of TGEV PUR46 Priorto infection TGEV PUR46 was treated with the indicated mucin concentrations for 30 min at room temperature After a 5 min adsorption time of the virus mucin mixture a plaque assay was performed Plaque reduction by mucin treatment was calculated out of 2 independent experiments with quadruplicates Standard deviations are indicated Significant differences are marked with an asterisk p value 0 05 Schwegmann We els et al Virology Journal 2011 8 435 Page 4 of 7 different mucin concentrations as described in the mate rial and methods section Figure 4 shows that TGEV infectivity was inhibited by the mucin in a concentration dependent manner Preincubation of the parental virus with 5 mg mucin ml reduced its infectivity by 62 when the virus adsorption time was 5 min Previous stu dies have shown that binding to these porcine intestinal mucins inhibited hemagglutination by TGEV 16 Thus sialic acid binding by the TGEV S protein appears to be inhibited by interaction with the tested mucin Conclusions From these results the picture arises that coronaviruses have developed different strategies to make use of the sialic acid binding activity These coronaviruses can be differentiated in three distinct groups TGEV PUR46 as a representative of the first group recognizes pAPN so efficiently that the contribution of the sialic acid binding activity to the infection of cultured cells has remained unrecognized so far Binding to pAPN is independent from sialic acids It even appears to be more efficient when less sialic acid is present on pAPN itself 5 Inter action with pAPN appears to be a slower process com pared to sialic acid binding Thus effects produced by sialic acid binding can be recognized at short adsorption times when pAPN binding is still incomplete Only by reducing the adsorption time we could show that the binding to sialoglycoconjugates on the cell surface increases the efficiency of TGEV PUR46 to infect cul tured cells For this strain the sialic acid binding activity may be required only to survive under unfavorable con ditions as encountered in the intestinal tract Interest ingly the Miller strain of TGEV did not show this significant difference in the infection efficiency between long and short adsorption times on neuraminidase trea ted cells that was observed with the Purdue strain There appear to be gradual differences in the impor tance of the sialic acid binding activity for different TGEV strains The Miller strain of TGEV resembles IBV in its dependence on the sialic acid binding activity These two viruses represent the second group of coro naviruses concerning their sialic acid binding activity Pre treatment of various cell types with neuraminidase has been shown to reduce the sensitivity to infection by different strains of IBV 14 15 A protein receptor has so far not been identified for this avian coronavirus The presence of such a receptor cannot be excluded The third group of coronaviruses with sialic acid binding activity is represented by viruses like bovine coronavirus BCoV and human coronavirus OC43 HCoV OC43 17 18 These viruses resemble influenza C virus not only because of their preference for N acetyl 9 O acetyl neuraminic acid but also because they contain an acety lesterase that functions as a receptor destroying enzyme Thepresenceofthisenzymeandthepreferencefora less common type of sialic acid are consistent with a higher affinity for the respective type of sialic acid On the other hand IBV which lacks a receptor destroying enzyme requires a larger amount of sialic acid on the cell surfaces than do Sendai virus or influenza viruses and thus has a lower affinity for sialic acid than the lat ter viruses which contain a neuraminidase as a receptor destroying enzyme 14 These three groups of corona viruses with sialic acid binding activity may represent different stages in an evolutionary process of using sialic acid for infection i acquisition of a sialic acid binding activity for increasing the efficiency of infection under unfavorable conditions TGEV Purdue ii modulation of the binding activity to exploit sialic acid as a general receptor determinant for attachment to target cells TGEV Miller IBV iii further increase of the attach ment efficiency by acquiring an enzyme that inactivates sialoglycoconjugates that prevent the spread of infection BCoV HCoV OC43 Coronaviruses are characterized by one of the highest recombination frequen- 1.請仔細(xì)閱讀文檔,確保文檔完整性,對于不預(yù)覽、不比對內(nèi)容而直接下載帶來的問題本站不予受理。
- 2.下載的文檔,不會出現(xiàn)我們的網(wǎng)址水印。
- 3、該文檔所得收入(下載+內(nèi)容+預(yù)覽)歸上傳者、原創(chuàng)作者;如果您是本文檔原作者,請點此認(rèn)領(lǐng)!既往收益都?xì)w您。
下載文檔到電腦,查找使用更方便
10 積分
下載 |
- 配套講稿:
如PPT文件的首頁顯示word圖標(biāo),表示該PPT已包含配套word講稿。雙擊word圖標(biāo)可打開word文檔。
- 特殊限制:
部分文檔作品中含有的國旗、國徽等圖片,僅作為作品整體效果示例展示,禁止商用。設(shè)計者僅對作品中獨創(chuàng)性部分享有著作權(quán)。
- 關(guān) 鍵 詞:
- 病毒,外文文獻(xiàn) 【病毒,外文文獻(xiàn)】2011 The sialic acid binding activity of protein facilitates infection by porcine transmissible 病毒
鏈接地址:http://m.italysoccerbets.com/p-7151479.html