scholarly journals Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

2001 ◽  
Vol 75 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
John T. Patton ◽  
Zenobia Taraporewala ◽  
Dayue Chen ◽  
Vladimir Chizhikov ◽  
Melinda Jones ◽  
...  
Keyword(s):  
Cell Culture ◽  
Consensus Sequence ◽  
Active Role ◽  
Serial Passage ◽  
Selective Advantage ◽  
Wild Type ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

scholarly journals A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

2003 ◽  
Vol 69 (4) ◽  
pp. 2052-2057 ◽  
Author(s):  
James C. Bull ◽  
H. C. J. Godfray ◽  
David R. O'Reilly
Keyword(s):  
Cell Culture ◽  
Population Genetics ◽  
Trichoplusia Ni ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Cell Infection ◽  
Occlusion Bodies ◽  
Budded Virus

ABSTRACT Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

scholarly journals Luxury at a Cost? Recombinant Mouse Hepatitis Viruses Expressing the Accessory Hemagglutinin Esterase Protein Display Reduced Fitness In Vitro

2005 ◽  
Vol 79 (24) ◽  
pp. 15054-15063 ◽  
Author(s):  
A. Lissenberg ◽  
M. M. Vrolijk ◽  
A. L. W. van Vliet ◽  
M. A. Langereis ◽  
J. D. F. de Groot-Mijnes ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis Virus ◽  
Natural Infection ◽  
Selective Advantage ◽  
Rna Recombination ◽  
Wild Type ◽  
Growth Properties ◽  
Group 2

ABSTRACT Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of “luxury” proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

scholarly journals The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

2020 ◽  
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Rajashri Sridharan ◽  
Christine M. O’Connor
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Latent Infection ◽  
Serial Passage ◽  
Viral Reactivation ◽  
Mrna Levels ◽  
Wild Type ◽  
Reading Frame ◽  
G Protein Coupled ◽  
Type Infection

AbstractThe ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

scholarly journals Identification of rocA, a Positive Regulator of covR Expression in the Group A Streptococcus

2003 ◽  
Vol 185 (10) ◽  
pp. 3081-3090 ◽  
Author(s):  
Indranil Biswas ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Single Copy ◽  
Wild Type ◽  
Global Regulator ◽  
Two Component System ◽  
Reading Frame ◽  
Two Component ◽  
Group A ◽  
Related Group ◽  
Hyaluronic Acid Capsule

ABSTRACT In the group A streptococcus (GAS; Streptococcus pyogenes), a two-component system known as CovRS (or CsrRS) regulates about 15% of the genes, including several important virulence factors like the hyaluronic acid capsule. Most of these genes, including covR itself, are negatively regulated by CovR. We have isolated two independent ISS1 insertions in an open reading frame (ORF) that increases CovR expression as measured by a Pcov-gusA reporter fusion in single copy in the GAS chromosome. This ORF, named rocA for “regulator of Cov,” activates covR transcription about threefold. As expected, a rocA mutant is mucoid and produces more transcript from the has promoter since this promoter is repressed by CovR. This effect is dependent on the presence of a wild-type covR gene. In contrast to its activation of Pcov, RocA negatively regulates its own expression. This autoregulation is not dependent on the presence of the covR gene. All the phenotypes of the rocA mutant were complemented by the presence of the rocA gene on a plasmid. The rocA gene is present in strains of all nine M serotypes of GAS tested and is absent from strains representing 11 other groups of streptococci and related bacteria, including strains of the closely related group C and G streptococci. It seems likely that rocA plays an important role in the pathogenesis of GAS since it affects expression of the global regulator CovR.

scholarly journals Transfection of exogenous rotavirus rearranged RNA segments in cells infected with a WT rotavirus results in subsequent gene rearrangements

2014 ◽  
Vol 95 (9) ◽  
pp. 2089-2098 ◽  
Author(s):  
Sarah Duponchel ◽  
Cécile Troupin ◽  
Lan Trang Vu ◽  
Aurélie Schnuriger ◽  
Germain Trugnan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Acute Gastroenteritis ◽  
Reverse Genetics ◽  
Gene Rearrangements ◽  
Recombinant Viruses ◽  
Bovine Strain ◽  
The Family ◽  
Viral Progeny ◽  
Group A Rotaviruses ◽  
Group A

Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.

scholarly journals The Genome Segments of a Group D Rotavirus Possess Group A-Like Conserved Termini but Encode Group-Specific Proteins

2010 ◽  
Vol 84 (19) ◽  
pp. 10254-10265 ◽  
Author(s):  
Eva Trojnar ◽  
Peter Otto ◽  
Bernhard Roth ◽  
Jochen Reetz ◽  
Reimar Johne
Keyword(s):  
Sequence Data ◽  
Open Reading Frames ◽  
Degenerate Primers ◽  
Nucleotide Sequence Data ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Specific Proteins ◽  
Group B ◽  
Group D

ABSTRACT Rotaviruses are a leading cause of viral acute gastroenteritis in humans and animals. They are grouped according to gene composition and antigenicity of VP6. Whereas group A, B, and C rotaviruses are found in humans and animals, group D rotaviruses have been exclusively detected in birds. Despite their broad distribution among chickens, no nucleotide sequence data exist so far. Here, the first complete genome sequence of a group D rotavirus (strain 05V0049) is presented, which was amplified using sequence-independent amplification strategies and degenerate primers. Open reading frames encoding homologues of rotavirus proteins VP1 to VP4, VP6, VP7, and NSP1 to NSP5 were identified. Amino acid sequence identities between the group D rotavirus and the group A, B, and C rotaviruses varied between 12.3% and 51.7%, 11.0% and 23.1%, and 9.5% and 46.9%, respectively. Segment 10 of the group D rotavirus has an additional open reading frame. Generally, phylogenetic analysis indicated a common evolution of group A, C, and D rotaviruses, separate from that of group B. However, the NSP4 sequence of group C has only very low identities in comparison with cogent sequences of all other groups. The avian group A NSP1 sequences are more closely related to those of group D than those of mammalian group A rotaviruses. Most interestingly, the nucleotide sequences at the termini of the 11 genome segments are identical between group D and group A rotaviruses. Further investigations should clarify whether these conserved structures allow an exchange of genome segments between group A and group D rotaviruses.

scholarly journals Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

2010 ◽  
Vol 84 (13) ◽  
pp. 6711-6719 ◽  
Author(s):  
Cécile Troupin ◽  
Axelle Dehée ◽  
Aurélie Schnuriger ◽  
Patrice Vende ◽  
Didier Poncet ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Helper Virus ◽  
Wild Type ◽  
Human Rotavirus ◽  
Protein Functions ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

scholarly journals Reduced Virulence of Group A Streptococcal Tn916 Mutants That Do Not Produce Streptolysin S

1998 ◽  
Vol 66 (4) ◽  
pp. 1671-1679 ◽  
Author(s):  
Stephen D. Betschel ◽  
Sergio M. Borgia ◽  
Neil L. Barg ◽  
Donald E. Low ◽  
Joyce C. S. De Azavedo
Keyword(s):  
Promoter Region ◽  
Open Reading Frame ◽  
Donor Strain ◽  
Group A Streptococci ◽  
Wild Type ◽  
Reading Frame ◽  
Streptolysin S ◽  
Streptolysin O ◽  
Group A ◽  
Type Strains

ABSTRACT Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn916insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn916 donor strain Enterococcus faecalis CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn916 insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated sagA, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus −1.16 g; P < 0.05) and had fewer necrotic lesions (0 versus 7; P = 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus −0.66 g; P< 0.05) and produced fewer necrotic lesions (1 versus 8;P = 0.001). Revertants of the mutants in which Tn916 had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn916 mutations, are markedly less virulent than their isogenic parents.

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

2008 ◽  
Vol 89 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Mark P. Zwart ◽  
Eloy Erro ◽  
Monique M. van Oers ◽  
J. Arjan G. M. de Visser ◽  
Just M. Vlak
Keyword(s):  
Cell Culture ◽  
Purifying Selection ◽  
Serial Passage ◽  
Autographa Californica ◽  
Homologous Region ◽  
Deletion Mutants ◽  
Wild Type ◽  
Insect Larvae ◽  
Selection For

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.

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