《【病毒外文文獻】2014 Regulation of coronaviral poly(A) tail length during infection is not coronavirus species- or host cell-specific》由會員分享，可在線閱讀，更多相關《【病毒外文文獻】2014 Regulation of coronaviral poly(A) tail length during infection is not coronavirus species- or host cell-specific（10頁珍藏版）》請在裝配圖網(wǎng)上搜索。

Regulation of coronaviral poly A tail length during infection is not coronavirus species or host cell specific Jui Hung Shien Yi Da Su Hung Yi Wu Received 16 March 2014 Accepted 4 July 2014 Published online 18 July 2014 C211 Springer Science Business Media New York 2014 Abstract It has been demonstrated that the length of the poly A tail in the bovine coronavirus BCoV which belongs to genus betacoronaviruses is regulated through out infection in human rectal tumor 18 HRT 18 cells and the length of the poly A tail is associated with the effi ciency of virus translation Here we examined whether the regulation of viral poly A tail length is cell type inde pendent and whether it is a common feature of coronavi ruses to assess the significance of the regulation By ligating head to tail viral RNA positive strands and sequencing we found that 1 the regulation pattern of coronaviral poly A tail length in BCoV infected hamster kidney 21 BHK 21 cells was similar to that in BCoV infected HRT 18 cells and 2 the poly A tail length of wild type avian infectious bronchitis virus IBV virulent strain IBV TW1 which is in the genus gammacoronavi ruses varied throughout infection in primary chicken embryo kidney CEK cells and in the tracheas of 1 day old chicks Interestingly the poly A tail length variation was similarly found in the avirulent IBV strain H120 in CEK cells although the overall poly A tail length was shorter for this virus The results suggest that the regulation of coronaviral poly A tail length during infection may be a common feature among coronaviruses and can occur in a noncancerous cell line BHK 21 cells primary cell culture CEK cells and living system chickens further rein forcing the biological significance of this regulation during coronavirus infection Keywords Bovine coronavirus C1 Polyadenylation C1 Avian infectious bronchitis virus C1 Poly A tail C1 Regulation Introduction The Coronavirinae which is subdivided into the alpha coronaviruses betacoronaviruses gammacoronaviruses and deltacoronaviruses belongs to the family Coronavir idae and the order Nidovirales The coronavirus genome contains a 5 0 cap 5 0 and 3 0 untranslated regions UTRs and a poly A tail 1 and therefore structurally is similar to eukaryotic messenger RNAs In bovine coronavirus BCoV which is in the genus betacoronaviruses the regulation of coronaviral ploy A tail length has been found during infection of human rectum tumor 18 HRT 18 cells 2 Specifically the viral poly A tail length in total viral RNA and subgenomic RNA 7 is relatively short 26 45 nucleotides in infected cells at 0 2 h postin fection hpi increases to peak length 65 nucleotides at 6 10 hpi and gradually decreases in size 30 45 nucleotides after 10 h of infection In addition the length of the coronaviral poly A tail is determined to be associated with the efficiency of virus translation 2 The cytoplasmic regulation of poly A tail length has also been well documented in certain mRNAs during the maturation of oocytes 3 8 and the regulated poly A tail length in these mRNAs is also linked to the regulation of translation Electronic supplementary material The online version of this article doi 10 1007 s11262 014 1103 7 contains supplementary material which is available to authorized users J H Shien C1 Y D Su Department of Veterinary Medicine Graduate Institute of Veterinary Pathobiology College of Veterinary Medicine National Chung Hsing University Taichung Taiwan ROC H Y Wu however most cell lines are derived from tumor cells which may have undergone mutations and preserve some aspects of disease 12 13 As a con sequence the biological function that is identified in these cell lines may not be applied to primary cells or living animals and therefore the identified function may not be significant 14 In the current study to determine whether the regulation of coronaviral poly A tail length is a com mon property among coronaviruses and can occur in non cancerous cells and animals IBV virulent strain TW1 IBV TW1 15 16 a causative pathogen in the world wide poultry industry which is in the genus gammacoro naviruses and is a phylogenetically distant coronavirus from BCoV was selected and tested In addition the IBV avirulent strain H120 which has been used in broilers to protect chickens against wild type wt IBV infection 17 was also employed to test whether the regulation was dif ferent between the avirulent and virulent strains of IBV We concluded that the regulation may be a common fea ture of coronaviruses and is not host cell specific Thus the regulated coronaviral poly A tail length may play an important role during coronavirus infection and the cells and animal species used in this study could be adequate candidates to approach the detailed mechanisms of the regulation Materials and methods Cells and viruses HRT 18 cells and Baby Hamster Kidney 21 BHK 21 cells were maintained in Dulbecco s modified Eagle s medium DMEM supplemented with 10 newborn calf serum in an incubator containing 5 CO 2 at 37 C176C The Mebus strain of BCoV was plaque purified three times and grown in a HRT 18 cell line as described previously 18 19 Primary cultures of chicken embryo kidney CEK cells were prepared from 18 day old chicken embryonic eggs and grown in minimum essential medium supple mented with 10 serum at 37 C176Cina5 CO 2 incubator and IBV TW1 15 16 and H120 Intervet UK were propagated in the allantoic cavity of 9 day old specific pathogen free SPF embryonic eggs Animal Health Research Institute Council of Agriculture Tamsui Tai wan The allantoic fluid of IBV TW1 or H120 was collected at 72 h postinoculation and used to infect CEK cells The supernatant collected at 48 hpi from infected CEK cells was used as the inoculum in the subsequent experiments Preparation of viral RNA To prepare viral RNA from BCoV infected BHK 21 cells confluent BHK 21 cells in a 35 mm diameter dish was infected with the Mebus strain of BCoV at a multiplicity of infection MOI of 1 PFU cell and total cellular RNA was extracted using TRIzol Invitrogen at different time points postinfection as indicated in the experiment To determine the poly A tail length in the inoculum of IBV TW1 and H120 viral RNA from purified IBV TW1 and H120 was extracted using TRIzol Invitrogen To prepare viral RNA from IBV TW1 or H120 infected CEK cells confluent CEK cells in 35 mm dishes were infected with inoculum of IBV TW1 or H120 at an MOI of 0 1 PFU cell and total cellular RNA was extracted using TRIzol Invitrogen To prepare viral RNA from the tracheas of IBV TW1 infected SPF chickens 18 1 day old SPF chickens were inoculated with 0 5 ml of IBV TW1 2 9 10 5 PFU ml via the con junctival and intranasal routes 20 The tracheas were then removed at the indicated time points and the viral RNA was extracted using TRIzol Invitrogen Chickens were maintained according to the guidelines established in the Guide for the Care and Use of Laboratory Animals prepared by the Committee for the Care and Use of Lab oratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council USA The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of National Chung Hsing University Taiwan Head to tail ligation of viral RNA RT PCR and sequencing A head to tail ligation method has been used to determine the lengths of coronaviral poly A tails 2 and the terminal features of influenza virus 21 Specifically 10 lgof extracted RNA in 25 ll of water 3 llof109 ligation buffer and 10 U of in 1 ll tobacco acid pyrophosphatase Epicenter were used to remove the 5 0 capped end of the viral RNA The decapped viral RNA was then extracted with phenol chloroform dissolved in 25 ll of water heat denatured at 95 C176C for 5 min and quick cooled for 1 min For head to tail ligation of decapped viral RNA 3 llof 109 ligase buffer and 2 U in 2 ll of T4 RNA ligase I New England Biolabs were then added and the reaction was incubated for 16 h at 16 C176C The head to tail ligated viral RNA was then extracted using phenol chloroform dissolved in water and quantitated For the RT reaction 384 Virus Genes 2014 49 383 392 123 1 lg of decapped and head to tailed RNA was used with SuperScript II reverse transcriptase Invitrogen which is able to transcribe poly A tails of longer than 100 nt with fidelity 22 23 and the resulting cDNA was used for PCR with AccuPrime Taq DNA polymerase Invitrogen To determine the length of the poly A tail of viral RNA extracted from BCoV infected BHK 21 cells the primer BCV29 54 was used for RT For the PCR 5 ll of the resulting cDNA mixture was used in a 50 ll PCR with primers BCV29 54 and BCV3UTR2 To examine the length of the poly A tails on the viral RNA that was extracted from IBV TW1 infected CEK cells H120 infected CEK cells and tracheas of IBV TW1 infected chicks the primer IBV5UTR was used for cDNA synthesis For PCR 5 ll of the resulting cDNA mixture was used in a 50 ll PCR with primers IBV5UTR and IBV3UTR The resulting 50 ll PCR mixture was heated to 94 C176C for 2 min then subjected to 34 cycles of 30 s at 94 C176C 30 s at 55 C176C and 30 s at 72 C176C The PCR product was directly sequenced to determine the length of the poly A tail The oligonucleotides used in this study are listed in Table S1 Western blot analysis BHK 21 cells in 35 mm dishes at 80 confluency 8 9 10 5 cells dish were infected with BCoV at an MOI of 1 PFU cell Cell lysates were harvested at different time points of infection electrophoresed through 12 SDS PAGE gels and electrotransferred to nitrocellulose membranes Amersham Biosciences BCoV nucleocapsid N proteins were detected using an antibody specific to the BCoV N protein as the primary antibody and goat anti mouse IgG conjugated to HRPO as the secondary antibody Jackson Laboratory The proteins detected were visual ized using Western Lightning TM Chemiluminescence Reagent Perkin Elmer NEL105 and X ray film Kodak Results Regulation of coronaviral poly A tail length in BHK 21 cells during BCoV infection A head to tail ligation method has been previously used to measure the lengths of poly A tails 2 21 and is depicted in Fig 1a Using this method we have demonstrated that the length of the coronaviral poly A tail is regulated during BCoV infection in HRT 18 cells 2 To determine whether the regulation of the coronaviral poly A tail length is host cell specific an inoculum with an infectious titer of 10 6 PFU ml BCoV and a poly A tail length of 45 nt 2 was used to infect freshly confluent BHK 21 cells with an MOI of 1 PFU cell Total cellular RNA was then extracted at the time points indicated in Fig 1b and used for head to tail ligation to measure the lengths of the poly A tails Since genome and subgenome of coronaviruses contain the same sequence of 3 0 UTR the sizes of poly A tail measured with this method reflect the lengths in the major population of total positive strand BCoV RNA including genomic RNA and subgenomic RNA As shown in Fig 1b the length of the RT PCR products lanes 5 13 which contained the sequences from BCoV 3 0 UTR poly A tail and BCoV 5 0 UTR varied and ranged from 200 to 250 base pairs bp After direct sequencing of the RT PCR products the lengths of the poly A tails represented in the major population of the total BCoV positive strand RNA were determined to be 33 nt 1 4 hpi 62 nt 8 hpi 51 nt 12 hpi 41 nt 18 24 hpi and 40 nt 48 72 hpi Fig 1c The poly A tail length was increased during early infection 0 8 hpi but gradually decreased after 8 h of infection throughout the 72 h period of infection The regulated pat tern of coronaviral poly A tail length in BCoV infected BHK 21 cells was similar to that in BCoV infected HRT 18 cells 2 After three rounds of independent experiments the amount of viral RNA at each time point was quantitated according to the intensity of the RT PCR products Fig 1b As shown in Fig 1d the amount of viral RNA increased markedly over time with the lengthening of poly A tail during the early infection suggesting that the efficiency of viral RNA synthesis may correlate with the poly A tail length To test further whether the increase of viral poly A tail length is also associated with the translation of coronavirus accumulation of the BCoV N protein was measured by Western blot analysis Fig 1e left panel As shown in Fig 1e right panel the synthesis efficiency of BCoV N protein was also significantly enhanced over time with the increase of poly A tail length during the early infection Taken together these results suggest that 1 the length of the poly A tail on the total positive strand BCoV RNA is also regulated during infection in BHK 21 cells demonstrating that this regulation is not host cell specific and 2 the increased efficiency of gene expression in the early infection of coronavirus may correlate with the lengthening of coronaviral poly A tail Coronaviral poly A tail length is regulated in primary CEK cells during IBV TW1 infection Immortalized cell lines have been used in viral studies for several decades because they replicate with no restriction and are readily available 11 Some of these cell lines were established from cancerous cells for example HRT 18 cells 24 which were used for the study of regulated coronaviral poly A tail length during BCoV infection 2 therefore they may still preserve certain features of the Virus Genes 2014 49 383 392 385 123 disease and have altered the normal biology of the cells in vivo In contrast primary cell cultures are physiologi cally similar to normal cells in vivo and may represent living systems To determine whether the regulation of coronaviral poly A tail also occurs in primary cell cultures and whether the regulation of viral poly A tail length is a Fig 1 Determination of coronaviral poly A tail length in BCoV infected BHK 21 cells a Strategy for determining coronaviral poly A tail length Total cellular RNA from virus infected cells was collected decapped and head to tail ligated The ligated viral RNA was used as template for RT with 5 0 UTR specific primer 2 and PCR was performed with 3 0 UTR specific primer 1 and primer 2 The synthesized RT PCR product contained the sequences from BCoV 3 0 UTR poly A tail and BCoV 5 0 UTR b The RT PCR products synthesized with RNA from BCoV inoculum lane 2 and with total cellular RNA collected from mock infected BHK 21 cells lane 3 and BCoV infected BHK 21 cells at different time points of infection lanes 5 13 by the method described in a c The length of the poly A tail as determined by sequencing the RT PCR products from samples obtained from b lane 2 and lanes 5 13 The length of poly A tail at 1 hpi was compared with that at different time points of infection for statistical analysis d Quantitation analysis of the RT PCR products shown in b lanes 5 13 e Left panel expression of BCoV N protein as analyzed by Western blot analysis Right panel quantitation analysis of BCoV N protein at different time points of infection M ds DNA size markers in nt pairs In inoculum Values in c e represent the mean SD of three individual experiments p 0 05 p 0 01 p 0 001 386 Virus Genes 2014 49 383 392 123 common feature among coronaviruses the IBV virulent strain IBV TW1 which is in the genus gammacoronavi ruses and is phylogenetically distant from BCoV was selected and tested in primary CEK cells We first deter mined the poly A tail length of the IBV TW1 that were used for inoculum For this the IBV TW1 prepared from the supernatant of IBV TW1 infected CEK cells was har vested at 48 hpi The viral RNA that was extracted from the pelleted IBV TW1 was decapped by tobacco acid pyrophosphatase and ligated head to tail by T4 RNA ligase I RT PCR was then performed using a primer set that specifically anneals to the 5 0 and 3 0 UTRs of the IBV TW1 genome as described in the Materials and Methods and the resulting RT PCR product Fig 2a left panel lane 2 was sequenced to determine the precise viral poly A tail lengths The results showed that the length of the poly A tail of IBV TW1 in the inoculum was 41 nt Fig 2a right panel To test whether the poly A tail length of IBV TW1 in CEK cells was regulated during infection the IBV TW1 inoculum was used to infect CEK cells with an MOI of 0 1 PFU cell Total cellular RNA was extracted at the time points indicated in Fig 2b and the length of the viral poly A tail was determined as described above The length of the RT PCR products ran ged from 250 to 300 bp was observed as shown in Fig 2b By direct sequencing of the RT PCR products the lengths of poly A were determined to be 36 57 38 37 36 36 and 32 nt at 1 2 8 12 24 36 and 48 hpi respectively Fig 2c suggesting the poly A tail length is regulated during IBV TW1 infection Fig 2 Poly A tail length of viral RNA from IBV TW1 infected CEK cells a Left panel RT PCR products synthesized with RNA from IBV TW1 inoculum lane 2 and with total cellular RNA collected from mock infected CEK cells lane 3 Right panel sequence of the poly A tail in the inoculum of IBV TW1 b The RT PCR product synthesized with total cellular RNA collected from IBV TW1 infected CEK cells at different time points of infection lanes 2 8 by the method described in Fig 1a except that the primers used were IBV5UTR for RT and IBV3UTR c The viral poly A tail lengths as determined by sequencing the RT PCR products that were obtained from b lanes 2 8 The length of poly A tail at 1 hpi was compared with that at different time points of infection for statistical analysis d Quantitation analysis of the RT PCR products shown in b M ds DNA size markers in nt pairs In inoculum Values in c and d represent the mean SD of three individual experiments p 0 05 p 0 01 Virus Genes 2014 49 383 392 387 123 in CEK cells By quantitating the intensity of the RT PCR products from Fig 2b it was found that the amount of viral RNA increased markedly over time with the lengthening of poly A tail during the early infection Fig 2d Thus these results suggest that 1 the regulation of coronaviral poly A tail length is not host cell specific or coronavirus species specific and 2 the increased efficiency of gene expression in the early infection of IBV TW1 may also correlate with the lengthening of coronaviral poly A tail in primary cell cultures The coronaviral poly A tail length is regulated in the tracheas of IBV TW1 infected chickens Thus far we have demonstrated that the poly A tail length in coronaviruses is regulated in a cancerous cell line HRT 18 cells 2 a noncancerous cell line BHK 21 cells Fig 1 and primary cell culture CEK cells Fig 2 To further investigate whether this feature also occurs in ani mals 1 day old SPF chickens were inoculated with a 0 5 ml inoculum containing an infectious titer of 2 9 10 5 PFU ml of IBV TW1 The tracheas were col lected from the chickens at the indicated time points in Fig 3a and RNA that was extracted from the tracheas was decapped and head to tail ligated RT PCR was carried out using a primer set as described above for determining poly A tail length of wild type IBV TW1 in CEK cells The lengths of the RT PCR products were varied throughout the 10 days of infection as shown in Fig 3a right panel The poly A tail length was then determined by direct sequencing of the RT PCR products As illustrated in Fig 3b the viral poly A tail length was 45 nt at 1 day postinfection dpi and then increased to 60 nt at 2 dpi After 2 days of infection the length of the poly A tail gradually decreased 51 and 47 nt at 3 and 5 dpi respectively Interestingly the poly A tail length was increased again after 5 days of infection 49 and 67 nt at 6 and 10 dpi respectively By quantitating the intensity of the RT PCR products from Fig 3a high level of the viral RNA was detected at 1 dpi Fig 3c however the level of viral RNA was decreased at 2 dpi and then increased markedly at 3 dpi The amount of viral RNA was maintained at a high level after 3 days of infection and then gradually decreased at 5 days of infection We suggested that the large