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• W2055463894 abstract "Article16 September 2004free access Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein Karen Kearney Karen Kearney Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, Bethesda, MD, USAPresent address: Department of Microbiology, University College Cork, Cork, Ireland Search for more papers by this author Dayue Chen Dayue ChenPresent address: Infectious Diseases Research, Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46285, USA Search for more papers by this author Zenobia F Taraporewala Zenobia F Taraporewala Search for more papers by this author Patrice Vende Patrice VendePresent address: Virologie Moléculaire et Structurale, Unité Mixte de Recherche, CNRS-INRA, 91198 Gif-sur-Yvette, France Search for more papers by this author Yasutaka Hoshino Yasutaka Hoshino Search for more papers by this author Maria Alejandra Tortorici Maria Alejandra Tortorici Search for more papers by this author Mario Barro Mario Barro Search for more papers by this author John T Patton Corresponding Author John T Patton Search for more papers by this author Karen Kearney Karen Kearney Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, Bethesda, MD, USAPresent address: Department of Microbiology, University College Cork, Cork, Ireland Search for more papers by this author Dayue Chen Dayue ChenPresent address: Infectious Diseases Research, Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46285, USA Search for more papers by this author Zenobia F Taraporewala Zenobia F Taraporewala Search for more papers by this author Patrice Vende Patrice VendePresent address: Virologie Moléculaire et Structurale, Unité Mixte de Recherche, CNRS-INRA, 91198 Gif-sur-Yvette, France Search for more papers by this author Yasutaka Hoshino Yasutaka Hoshino Search for more papers by this author Maria Alejandra Tortorici Maria Alejandra Tortorici Search for more papers by this author Mario Barro Mario Barro Search for more papers by this author John T Patton Corresponding Author John T Patton Search for more papers by this author Author Information Karen Kearney1, Dayue Chen, Zenobia F Taraporewala, Patrice Vende, Yasutaka Hoshino, Maria Alejandra Tortorici, Mario Barro and John T Patton 1Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, Bethesda, MD, USA *Corresponding author. Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, 50 South Drive MSC 8026, NIH, Bethesda, MD 20892, USA. Tel.: +1 301 496 5227; Fax: +1 301 496 8312; E-mail: [email protected] The EMBO Journal (2004)23:4072-4081https://doi.org/10.1038/sj.emboj.7600408 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3′-consensus sequence (3′CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3′CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3′-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0–5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3′CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines. Introduction Rotavirus, a member of the family Reoviridae, is the primary cause of acute dehydrating diarrhea in infants and young children worldwide (Parashar et al, 2003). The icosahedral core of the triple-layered rotavirus particle contains the 11 segments of double-stranded (ds)RNA that make up the viral genome (Prasad et al, 1988). The segmented nature of the genome demands that the viral RNA-dependent RNA polymerase (RdRP) recognize and employ multiple viral mRNAs as templates for dsRNA synthesis. The synthesis of the dsRNA genome is a process that is coordinated with the packaging of the template mRNAs into previrion cores (Patton and Spencer, 2000). The genome segments are distributed in an orderly manner within the core, each associated with one of the 12 vertices (Prasad et al, 1996). Along with a genome segment, a copy of the viral RdRP (VP1) and mRNA-capping enzyme (VP3) are also associated with each vertex (Estes, 2001). The RdRPs transcribe the genome segments to produce mRNAs that are extruded from the core through channels present at the vertices (Figure 1) (Lawton et al, 1997). Figure 1.Importance of the 3′CS to rotavirus RNA replication and gene expression. The 11 dsRNA genome segments (upper left) contain highly conserved sequences at their termini, including the 3′CS (red) at the 3′-end of the (+) strand. Activation of the RdRPs within double-layered particles yields (+) RNAs, which are extruded through channels located at the vertices. During gene expression, NSP3 dimers simultaneously interact with the last four to five nucleotides of the 3′CS and with eukaryotic initiation factors, causing polysome circularization. During RNA replication, the RdRP recognizes signals in the 3′CS and upstream region of the (+) RNA. Recognition is followed by the formation a (−) strand initiation complex, an event that requires the 3′CC of the (+) strand template, the RdRP and core scaffold protein VP2, and GTP. Initiation and (−) strand synthesis are associated with the packaging of (+) RNAs into cores. The assembly of a VP6 layer around the core produces a double-layered particle. Download figure Download PowerPoint Transcription of the rotavirus dsRNA genome produces 11 mostly monocistronic mRNAs that contain 5′-methylated caps but lack 3′-poly(A) tails (Estes, 2001). Sequencing has indicated that the viral mRNAs of the most common group of rotaviruses (group A) begin with 5′-GGC(U/A)7-3′ and, with only rare exception, end with the 3′-consensus sequence (3′CS), 5′-UGUGACC-3′ (Figure 1) (Mitchell and Both, 1990a; Desselberger and McCrae, 1994). The conservation of the 3′CS suggests that it constitutes part of one or more cis-acting signals for processes that all the viral mRNAs undergo during virus replication. Studies on the function of the 3′CS bear this out, having indicated that the element contains information for at least three functions—(i) polymerase recognition: Electrophoretic mobility shift assays (EMSAs) have shown that the 3′CS contains sufficient information to allow the rotavirus RdRP to specifically recognize viral mRNAs. However, the 3′CS represents only a part of the recognition signal since the RdRP can bind specifically to viral mRNAs even upon deletion of the 3′CS (Tortorici et al, 2003); (ii) promoter activity: Template-dependent replication assays have demonstrated that deletion of the 3′CS prevents viral mRNAs from serving as templates for (−) strand synthesis (Patton et al, 1996). Such assays have also indicated that the 3′CC of the mRNAs is of critical importance for dsRNA synthesis, while the remaining residues of the 3′CS are of variable importance (Chen et al, 2001); (iii) efficient translation: The last four to five nucleotides of the 3′CS, (U)GACC, are specifically recognized by dimers of NSP3, a viral nonstructural protein which also has affinity for the cap-associated eukaryotic initiation factor, eIF4GI (Poncet et al, 1994; Piron et al, 1998; Deo et al, 2002; Groft and Burley, 2002). By binding simultaneously to both eIF4GI and the 3′-terminal (U)GACC sequence, NSP3 acts as a translation enhancer that upregulates viral gene expression by promoting the circularization of viral mRNAs in polysomes (Figure 1) (Chizhikov and Patton, 2000; Vende et al, 2000). In this regard, NSP3 operates as a rotavirus-specific surrogate of the eukaryotic poly-A-binding protein (PABP; Kahvejian et al, 2001). Given the multiple functions for the 3′CS, any mutation of the element could be predicted to lead to a loss of virus viability. However, our analysis of a genome segment of rhesus rotavirus (RRV), a strain which forms the basis of the tetravalent live-attenuated human RotaShield vaccine (Wyeth-Lederle Vaccines and Pediatrics) (Kapikian et al, 1996), reveals that this is not the case. Specifically, we have found that the 3′CS of the gene 5 mRNA (g5(+) RNA) of RRV undergoes rapid and radical change, but only upon serial passage of the virus in highly permissive cells at high multiplicity of infection (MOI). Such changes were not observed upon serial passage in highly permissive cells at low MOI or in less permissive cells. The product of the rotavirus g5(+) RNA is NSP1, a nonstructural protein whose interaction with the cytoplasmic resident protein, interferon (IFN) regulatory factor 3 (IRF3) (Graff et al, 2002), promotes cell-to-cell spread of the virus (Barro and Patton, 2004). This interaction suppresses the translocation of IRF3 to the nucleus, where otherwise the factor activates genes expressing IFN and induces the establishment of the antiviral state (Hiscott, 1999; Barro and Patton, 2004). Consistent with the importance of NSP1 in promoting virus spread is the observation that genetic changes to the rotavirus g5(+) RNA which truncate the NSP1 open reading frame (ORF) yield viruses with small plaque phenotype (Patton et al, 2001). We determined that mutation of the 3′CS has two overall effects on the RRV g5(+) RNA, increasing its efficiency as a replication template for g5 dsRNA and decreasing its efficiency as a translation template for NSP1. The latter appears to be tolerated under conditions of high MOI passage in highly permissive cells because of the diminished need for the viral protein (NSP1) promoting cell-to-cell spread. Our analysis redefines the essential 3′-terminal sequence required for dsRNA synthesis as URN0–5CC and indicates that the prototypic 3′CS represents a suboptimal promoter for RNA synthesis whose sequence is largely retained due to the role of the element as a translation enhancer. This study illustrates the importance of host cell line and passage conditions as variables affecting the phenotype of progeny viruses. These results also suggest that the efficacy of live-attenuated rotavirus vaccines may be impacted by the methods used in propagating the virus strains included in the vaccines. Results 3′-Sequence heterogeneity of g5(+) RNA of RRV Previous studies have shown that the g5(+) RNAs of simian SA11 and SA11-4F rotaviruses grown in the highly permissive MA104 cell line end with the mutant sequence, UGUGAaCC, due to the insertion of an A residue (lower case) into the 3′CS, UGUGACC (Mitchell and Both, 1990b; Patton et al, 2001). Sequencing of a large number of cDNAs generated by RACE confirmed the invariant nature of the mutant g5(+) 3′CS in laboratory stocks of SA11-4F (Table I). Table 1. 3′-Terminal sequences of gene 5 (+)RNAs of rotavirus propagated in MA104 cells Strain 3′-sequence Clones RRV (rhesus) UGccuCCa 6 RRV (rhesus) UGUuuCC 5 RRV (rhesus) UGcuuCC 3 RRV (rhesus) UGUuauCC 3 RRV (rhesus) UGauuCC 2 RRV (rhesus) UGUAaCC 1 RRV (rhesus) UGUugCC 1 RRV (rhesus) UGUcuCC 1 SA11-4F (simianb) UGUGAaCC 30 UK (bovine) UGUGACC 20, 37c a For this and subsequent tables, wild-type and mutant sequences are shown in upper and underlined lower case, respectively. b Gene 4 of SA11-4F is bovine rotavirus-like in origin, other genes are of simian SA11 origin. c Pools originated from separate sources. To examine whether the mutant g5(+) 3′CS characteristic of SA11 virus was common to other primate strains of rotavirus, cDNA clones of the g5 dsRNA recovered from laboratory stocks of RRV grown in MA104 cells were generated by RACE. Sequencing showed that the RRV g5(+) RNAs ended with neither the prototypic 3′CS nor the mutant g5(+) 3′CS of SA11 viruses (Table I). Instead, the RRV g5(+) 3′-sequences were heterogenous in nature, most differing by two or three residues from the 3′CS due to base substitutions at the −3 (A → U or G), −4 (G → U, C or A), and, in some cases, −5 (U → C or A) positions. Although the majority of the g5(+) 3′-sequences retained the heptamer length 3′CS, an insertional mutation caused the 3′CS to become octameric in some cases, that is, UGUuauCC. Notably, the terminal CC (−1, −2) and upstream UG (−6, −5) of the 3′CS were retained in all the RRV g5(+) RNAs. Unlike the g5(+) RNAs of SA11, SA11-4F and RRV, RACE analysis showed that all g5(+) RNAs contained in two independent lots of UK bovine rotavirus grown in MA104 cells ended with the 3′CS (Table I). Plaque isolation of RRV g5 variants To determine whether RRV variants with mutant g5(+) 3′CS could be clonally isolated, RRV grown in MA104 cells was subjected to triple-plaque-to-plaque purification. Afterwards, the plaque-purified viruses from the second and third cycles were amplified once at low MOI to generate dsRNAs for RACE analysis. Sequencing led to the identification of five unique variants, characterized largely by the substitution of residues at the −3, −4, and, less frequently, −5 position of the 3′CS with U (Table II). Little or no heterogeneity was detected for the g5(+) 3′-sequences of the variants RRVg5-auu, -cuu, -u, and -uu, recovered after the second and third rounds of plaque purification. The predominant g5(+) 3′-sequence of the variant RRVg5-u contained not only a base substitution but also a deletion. Thus, virus with g5 dsRNAs ending with a hexameric sequence instead of the heptameric 3′CS is viable. Analysis of the triple-plaque-purified variant RRVg5-ccuu gave results suggesting that its g5(+) 3′-sequence was not genetically stable, shifting between the octamer UGccuuCC and the tetramer UGCC (Table II). The UGCC sequence is significant as it is similar to the conserved sequence, (A/U)GCC, found at the 3′-end of the (−) strand of all rotavirus RNAs (3,16). The conservation of the (A/U)GCC sequence and its location at the 3′-end of the (−) strand of rotavirus RNAs indicates that it forms all or part of the transcription promoter for (+) strand synthesis. RACE analysis of the plaque-purified variant RRV-uu revealed that its g9(+) RNA ended with the prototypic 3′CS (data not shown). This finding indicates that mutation of the g5(+) 3′CS can occur in the absence of changes to the 3′-ends of the other viral (+) RNAs. Table 2. 3′-Sequence of g5(+) RNA of RRV variants isolated by triple plaque-to-plaque purification on MA014 cellsa Cycle two Cycle three Variant 3′-end Clones 3′-end Clones RRVg5-u UGUuCC 14 UGUuCC 12 UGUuuCC 1 RRVg5-uu UGUuuCC 6 UGUuuCC 9 UGUuCC 1 UGUuCC 1 UGUuuuCC 1 RRVg5-auu UGauuCC 9 UGauuCC 17 UGauCC 1 RRVg5-cuu UGcuuCC 13 UGcuuCC 14 RRVg5-ccuu UGccuuCC 7 UGccuuCC 7 UG–––CC 3 UG–––CC 5 UGccuCC 1 a GenBank accession numbers for g5(+) RNAs: RRVg5-cs, AY117048; RRVg5-u, AY117052; RRVg5-uu, AY117053; RRVg5-auu, AY117049; RRVg5-cuu, AY117051; RRVg5-ccuu, AY117050. Plaque isolation of a variant from a pool of DS1xRRV grown in rhesus fetal lung FRhL2 cells revealed that viruses with mutant g5(+) 3′-ends could also be isolated, which contained other than just RRV-specific genes. The reassortant virus DS1xRRV contains 10 genome segments from RRV and one segment (gene 9) from the human strain DS1 (Midthun and Kapkian, 1996). The g5(+) RNA of the variant, DS1xRRVg5-uu, was like that of RRVg5-uu, ending with the sequence UGUuuCC (Table II). One-step growth curves revealed no differences in virus production by DS1xRRVg5-uu and DS1xRRV containing g5(+) RNA with the prototypic 3′CS (DS1xRRVg5-cs) in MA104 cells (data not shown). Full-length cDNA clones were prepared from the g5 dsRNAs of DS1xRRV and RRV variants and their corresponding wild-type viruses, that is, DS1xRRVg5-cs and RRVg5-cs, respectively. Sequencing revealed that the g5(+) RNAs of DS1xRRVg5-uu and DS1xRRVg5-cs were identical, except for the expected 3′-terminal variations. Similarly, the sequences of the g5(+) RNAs of the RRV variants were identical to that of RRVg5-cs, except for the expected differences in their 3′-ends, and in some cases the replacement of nucleotide G178 in the RRVg5-cs sequence with a T in the variant sequences. When present, the G178>T change resulted in the conservative amino-acid substitution V50>L in the g5 product, NSP1. RACE analysis indicated that the sequence 5′-A8CGCC-3′ was present at the 3′-ends of the (−) strand of the g5 dsRNAs recovered from the RRV variants and from RRVg5-cs. Hence, the portions of the g5 dsRNAs predicted to be involved in initiation of transcription (i.e., (+) strand synthesis) were the same for the variant and wild-type viruses (Figure 1). Electrophoretic analysis revealed no differences in the sizes of any of the dsRNA genome segments among the RRV and DS1xRRV isolates (data not shown). Although their g5 dsRNAs lacked the 3′CS, the variant and wild-type viruses were able to grow to similar high titers (⩾108 plaque-forming units (PFU)/ml) in MA104 cells. Impact of cell passage on 3′-sequence variation Although the g5(+) 3′-ends of RRV grown in MA104 cells were variable and atypical in sequence (Table I), RACE analysis revealed that g5(+) RNAs of plaque-purified RRV serially passaged seven times in FRhL2 cells ended exclusively with the 3′CS (Table III). To explore the possibility that growth of RRV in MA104 cells promoted deviation of the g5(+) 3′CS, FRhL2-derived RRV was passaged serially in MA104 cells using low dilutions of infected cell lysates as inocula (initial MOI >10). RACE revealed that, even after a single passage in MA104 cells, g5(+) RNAs which lacked the 3′CS were present (Table III). By the third passage, 50% of the g5(+) RNAs lacked the 3′CS and, by the fifth passage, no g5(+) RNAs were detected that contained the 3′CS. Thus, serial passage of RRV at high MOI in MA104 cells strongly favored the production of virus lacking the g5(+) 3′CS. Table 3. Generation of RRV gene 5 variants by serial passage in MA104 cells MA104 cells (clones) High MOI Low moi FRhL2 cells (48) Pass 1 (27) Pass 2 (28) Pass 3 (40) Pass 5 (36) Pass 5 (31) UGUGACC 48 UGUGACC 23 UGUGACC 18 UGUGACC 19 UGUuauCC 24 UGUGACC 29 UGUuuCC 2 UGUu-CC 4 UGUu-CC 8 UGUu-CC 6 UGUuuCC 1 UGUu-CC 2 UGUuuCC 3 UGUuuCC 7 UGUuuCC 4 UGUugCC 1 UGUuACC 1 UGUuauCC 2 UGcu-CC 1 UGUuauCC 1 UGccuCC 2 UGU–CC 1 UGUuuuuCC 1 UGUauuCC 1 UGUGAaCC 1 MA104 cells (clones) Low moi Inspection of RRV passaged serially at high MOI revealed that the initial base substitutions at the g5(+) 3′-end were not random, but mostly involved replacement of A(−3) and/or G(−4) with U (Table III, pass 1 and 2). Both deletion and insertion of residues between −3 and −4 were also events observed early in the passage of RRV. Interestingly, the g5(+) 3′-end UGUuauCC that predominated in the pass-5 RRV pool (24/36) was not the predominant 3′-end of any earlier virus passage. This suggests that, although other variant 3′-ends (e.g., UGUGuuCC) may have evolved more readily during serial passage, stronger selective pressure existed favoring utilization and retention of the UGUuauCC end. Analysis of RRV serially passaged 50 times at high MOI in MA104 cells showed that the g5(+) 3′-ends were still somewhat heterogenous in nature (Table IV). However, the predominant g5(+) 3′-end UGUugCC of the pass 50 pool was unlike the predominant 3′-end of the pass 5 pool (Table III), instead showing similarity to the transcription-promoter sequence (A/U)GCC found at the 3′-end of rotavirus (−) sense RNAs (Figure 1). The UGUugCC end was not detected in any of the pass 1–5 pools of RRV (Table III), suggesting that it may have evolved by a multi-step process whereby variant g5(+) 3′-ends underwent successive rounds of mutation. The second most prevalent g5(+) 3′-end in the pass 50 pool was UGUGAaCC, and thus identical to the g5(+) 3′-ends of virus strains SA11, K9, and KU (Table VI). Table 4. Effect of cell line on the variation of the 3′-end of RRV g5(+) RNA Virus Cell type Passagea 3′-sequence Clones RRVb AGMK 50 UGcugCC 46 UGUGAaCC 5 UGUugCC 2 MA104 50 UGUugCC 32 UGUGAaCC 8 UGUG-CC 2 HCT 15 UGUGACC 7 Vero 10 UGUauCC 5 UGUuauCC 3 UGUuuCC 1 UGUu-CC 1 UGcuuCC 1 DS1xRRVg5-cs MA104 10 UGUuCC 11 UGUGACC 10 UGUGAuCC 8 UGUuuCC 5 UGcuuCC 1 aGcuuCC 1 SA11-4F MA104 10 UGUGACC 17 a Virus was serially passaged the indicated times using 1:3 dilutions of infected cell lysate to fresh media as inoculum. b RRV prepared in FRhL2 cells (see Table III). In contrast to the extensive mutation of the 3′CS that occurred upon serial passage of the virus at high MOI, the g5(+) RNAs of RRV recovered after five cycles of serial passage at high dilution (1:4200) in MA104 cells (initial MOI <0.1) terminated almost exclusively with the 3′CS (29 3′CS/32 total ends) (Table III). The differences in results obtained upon serial passage of RRV at low and high dilutions in MA104 cells indicates that MOI is a factor affecting the extent of change to the g5(+) 3′-end. Notably, at lower MOI where complete infection of the MA104 culture requires cell-to-cell spread, the g5(+) RNA retains the 3′CS. At higher MOI, where all cells in the culture are initially infected, negating the need for cell-to-cell spread, the g5(+) 3′-end undergoes rapid and extensive change. Genetic instability was also seen for the g5(+) 3′-end of DS1xRRV upon serial passage at low dilution (initial MOI >10) in MA104 cells. Specifically, 10 rounds of such passage yielded virus with g5(+) RNAs that ended predominantly with other than the 3′CS (10 3′CS/46 total ends) (Table IV). However, similar high MOI serial passage of SA11-4F in MA104 cells did not result in changes to its g5(+) 3′ sequence (Table IV). Hence, the potential for hypervariability of the g5(+) 3′-end is a characteristic of some but not all strains of rotavirus, and only some genes of RRV are required for the hypervariable phenotype. Impact of cell line on 3′-sequence variation The impact of cell line on mutation of the g5(+) 3′CS was further examined by serial passage of FRhL2-derived RRV at low dilution in primary or secondary African green monkey AGMK cells, human ileocecal adenocarcinoma HCT-8, and African green monkey Vero cells. A number of these (i.e., AGMK, FRhL2 and Vero) have been employed in the production of live-attenuated human rotavirus vaccines (Midthun and Kapikian, 1996; Bernstein et al, 1998). The analysis showed that the g5(+) RNAs of RRV passaged 50 times in AGMK cells ended not with the 3′CS, but predominantly with the 3′-sequence UGcugCC (Table IV). This end differs by only a single pyrimidine substitution at the −5 position from the predominant 3′-end detected upon passage of RRV 50 times in MA104 cells (UGcugCC versus UGUugCC), suggesting similarities in mutation rates, types of mutations, and selective pressures for the 3′-end of the RRV g5 RNA in both cell types. This suggestion is further supported by the observation that the second most common g5(+) 3′-ends detected in the 50-pass RRV pools from MA104 and AGMK cells were identical: UGUGAaCC (Table IV). Analysis of FRhL2-derived RRV passed 10 times in Vero cells at low dilution showed that this cell line, like the AGMK and MA104 lines, promoted deviation of the g5 3′CS (Table IV). In contrast, the g5 RNAs of RRV passaged even 15 times in the HCT-8 cells retained the prototypic 3′CS, a result reminiscent of passage of RRV in FRhL2 cells (Table III), which caused little or no deviation of the 3′CS. Importantly, AGMK, MA104, and Vero cells support the high titer growth of RRV, yielding infected cell lysates that are typically ⩾108 PFU/ml. In contrast, RRV titers achieved in FRhL2 cells are 1–2 logs lower. Likewise, HCT-8 cells are less efficient in supporting RRV growth, and, in fact, the virus is lost upon multiple rounds of passage in this cell line. Thus, hypervariability of the g5(+) 3′-end of RRV is a characteristic that can be correlated with serial passage of the virus in cell lines supporting high titer growth. Owing to their ability to support high titer growth, serial passage of RRV in AGMK, MA104, and Vero cells even at a the same low dilution (1:3) used in serial passage of the virus in FRhL2 and HCT-8 cells means that the AGMK, MA104, and Vero cells are likely infected at much higher MOI at each cycle than the FRhL2 and HCT-8 cells. As a consequence, complete infection of the FRhL2 and HCT-8 cell cultures is likely to be more dependent upon the efficient cell-to-cell spread of the serially passaged virus than is required for complete infection of the AGMK, MA104, and Vero cell cultures. 3′-Sequence of g5 RNAs of RRV vaccine strains Rotashield, the RRV-based tetravalent vaccine, was found to be highly efficacious in protecting against the four major rotavirus serotypes (G1–G4) that cause severe diarrhea in humans (Kapikian et al, 1996). The G1, G2, and G4 components of the vaccine were reassortant viruses containing a single gene (VP7) from human rotaviruses and 10 genes including g5 from RRV. RRV itself served as the G3 component (Kapikian et al, 1985; Midthun and Kapikian, 1996). VP7 is an outer capsid protein of rotavirus which stimulates protection in vaccines by inducing anti-VP7-neutralizing antibodies. The virus components of the vaccine were prepared by passage six to seven times in AGMK cells, followed by passage seven (RRV), 11 (DS1xRRV), 13 (ST3xRRV), or 16 (DxRRV) times in FRhL2 cells (Midthun and Kapikian, 1996). To gain further insight into the impact of the FRhL2 cell line on RRV g5(+) 3′CS variation, we analyzed the g5(+) 3′-ends of viruses contained in the vaccine. The analysis showed that, despite passage of the DxRRV, DS1xRRV, and RRV vaccine components up to 16 times in FRhL2 cells, these viruses contained few if any RRV g5(+) RNAs that lacked the 3′CS (Table V). Although significantly greater numbers of g5(+) RNAs lacking the 3′CS were present for the ST3xRRV component, the majority of the RNAs possessed the 3′CS. Overall, these results suggested that serial passage of the vaccine components in FRhL2 cells favored retention of the RRV g5(+) 3′CS. Table 5. 3′-Sequences of g5(+) RNAs of rotavirus vaccine strains propagated in FRhL2 cells RRV-based vaccine component (passage cycles in FRhL2 cells) DxRRV-G1 (16) DS1xRRV-G2 (11) RRV-G3 (7) ST3xRRV-G4 (13) UGUGACC 22 UGUGACC 21 UGUGACC 23 UGUGACC 14 UGUuuCC 1 UGUu-CC 1 UGUauCC 4 UGacuCC 1 UGUuauCC 1 UGUu-CC 3 UGUuuCC 1 UGUGAuCC 1 UK-based vaccine component UK-G3 (7) UGUGACC 48 3′-Motif necessary for genome replication The results presented above indicate that RRV remains viable even upon significant deviation of the g5(+) 3′CS. Considered en toto (see Tables I, II, III, IV and V), the data reveal that any g5(+) RNA can function as a template for dsRNA synthesis in vivo as long as it ends with the motif, UGN0–5CC (N=any nucleotide) (Table VI). Although there are several other rotaviruses with g5(+) 3′-ends that deviate from the 3′CS (e.g., K9, KU; Okada et al, 1999), their atypical g5(+) 3′-ends do not exhibit the extreme sequence hypervariability detected for RRV (Table VI). Table 6. Common 3′-motif for group A rotavirus (+) RNAs 3′-consensus sequence for RRV gene 5 UGN0–5CC 3′-ends of other group A viruses: SA11 gene 2 (VP2) UaUGACC K9 gene 5 (NSP1) UGUGAaCC KU gene 5 (NSP1) UGUGAaCC SA11 gene 5 (NSP1) UGUGAaCC SA11 gene 7 (NSP3) UGUGgCC Common 3′-motif URN0–5CC Common 3′-motif excluding gene 5 URUGRCC aR=ATP, GTP; N=ATP, CTP, GTP, UTP. Deviations in the 3′CS have also been noted for rotavirus genes, which, unlike g5, encode proteins that are essential for virus replication (Table VI). In particular, we confirmed by RACE the earlier finding that SA11 g2(+) (Mitchell and Both, 1990a) and g7(+) (Both et al, 1984) RNAs have atypical 3′-ends, resulting from the purine (R) substitutions G(−6) → A and A(−3) → G, respectively. The G(−6) → A deviation in the SA11 g2(+) 3′-end corresponds to one of the few residues of the 3′-end of the RRV g5(+) RNA noted for its absolute degree of conservation (UGN0–5CC). Though usually a G, the presence of the A at the −6 position of SA11 g2(+) RNA suggests that any purine at this position is sufficient to satisfy the requirements for virus viability (Table VI). The A(−3) → G deviation in the SA11 g7(+) 3′CS falls within the NSP3-recognition signal" @default.
• W2055463894 created "2016-06-24" @default.
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• W2055463894 date "2004-09-16" @default.
• W2055463894 modified "2023-09-27" @default.
• W2055463894 title "Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein" @default.
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