scholarly journals An alternative to the adenovirus inverted terminal repeat sequence increases the viral genome replication rate and provides a selective advantage in vitro

2014 ◽  
Vol 95 (7) ◽  
pp. 1574-1584 ◽  
Author(s):  
Kerstin Wunderlich ◽  
Esmeralda van der Helm ◽  
Dirk Spek ◽  
Mark Vermeulen ◽  
Adile Gecgel ◽  
...  
Keyword(s):  
Human Adenovirus ◽  
Selective Advantage ◽  
Adenoviral Vectors ◽  
Repeat Sequence ◽  
Inverted Terminal Repeat ◽  
Genome Replication ◽  
Inverted Terminal Repeat Sequence ◽  
Viral Genome Replication ◽  
Terminal Repeat Sequence

During the development of human adenovirus 35-derived replication-incompetent (rAd35) vaccine vectors for prevention of infectious diseases, we detected mutations in the terminal 8 nt of the inverted terminal repeats (ITRs) of rAd35. The switch from the plasmid-encoded sequence 5′-CATCATCA-3′ to the alternative sequence 5′-CTATCTAT-3′ in the ITRs was found to be a general in vitro propagation phenomenon, as shown for several vectors carrying different transgenes or being derived from different adenovirus serotypes. In each tested case, the plasmid-encoded ITR sequence changed to exactly the same alternative ITR sequence, 5′-CTATCTAT-3′. The outgrowth of this alternative ITR version should result from a growth advantage conferred by the alternative ITR sequence. Indeed, replication kinetics studies of rAd35 harbouring either the original or alternative ITR sequence confirmed an increase in replication speed for rAd35 vectors with the alternative ITR sequence. These findings can be applied to generate recombinant adenoviral vectors harbouring the alternative ITR sequence, which will facilitate the generation of genetically homogeneous seed virus batches. Moreover, vector production may be accelerated by taking advantage of the observed improved replication kinetics associated with the alternative ITR sequence.

scholarly journals Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression

2020 ◽  
Vol 31 (3-4) ◽  
pp. 151-162 ◽  
Author(s):  
Lauriel F. Earley ◽  
Laura M. Conatser ◽  
Victoria M. Lue ◽  
Amanda L. Dobbins ◽  
Chengwen Li ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Repeat Sequence ◽  
Terminal Repeat ◽  
Inverted Terminal Repeat ◽  
Adeno Associated Virus ◽  
Inverted Terminal Repeat Sequence ◽  
Virus Serotype ◽  
Terminal Repeat Sequence

Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata

Gene ◽  
1980 ◽  
Vol 10 (4) ◽  
pp. 301-306 ◽  
Author(s):  
Oka Yoshio ◽  
Shiota Susumu ◽  
Nakai Sumiko ◽  
Nishida Yasuyoshi ◽  
Okubo Shunzo
Keyword(s):  
Repeat Sequence ◽  
Terminal Repeat ◽  
Inverted Terminal Repeat ◽  
Inverted Terminal Repeat Sequence ◽  
Terminal Repeat Sequence

scholarly journals Adenovirus Late-Phase Infection Is Controlled by a Novel L4 Promoter

2010 ◽  
Vol 84 (14) ◽  
pp. 7096-7104 ◽  
Author(s):  
Susan J. Morris ◽  
Gillian E. Scott ◽  
Keith N. Leppard
Keyword(s):  
Gene Expression ◽  
Intermediate Phase ◽  
Expression Patterns ◽  
Late Phase ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Late Gene ◽  
Genome Replication ◽  
Late Gene Expression ◽  
Viral Genome Replication

ABSTRACT During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

scholarly journals Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

1987 ◽  
Vol 7 (3) ◽  
pp. 1063-1069
Author(s):  
M B Vasudevachari ◽  
V Natarajan ◽  
N P Salzman
Keyword(s):  
Rna Synthesis ◽  
Chloramphenicol Acetyltransferase ◽  
Repeat Sequence ◽  
Early Gene ◽  
Inverted Terminal Repeat ◽  
Adenovirus E1a ◽  
293 Cells ◽  
Inverted Terminal Repeat Sequence ◽  
Inverted Terminal Repeats ◽  
Terminal Repeats

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

scholarly journals Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Paula F. Zamora ◽  
Liya Hu ◽  
Jonathan J. Knowlton ◽  
Roni M. Lahr ◽  
Rodolfo A. Moreno ◽  
...  
Keyword(s):  
Virus Replication ◽  
Viral Genome ◽  
Nonstructural Protein ◽  
Dsrna Virus ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Content Type ◽  
The Family ◽  
Viral Genome Replication

ABSTRACTViral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, usingin vitroand cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCEFollowing infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the familyReoviridaeencode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the familyReoviridaediverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Usingin vitroand cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the familyReoviridaeassort and replicate their genomes.

scholarly journals Effects of Long Terminal Repeat Sequence Variation on Equine Infectious Anemia Virus Replication in Vitro and in Vivo

Virology ◽  
1999 ◽  
Vol 263 (2) ◽  
pp. 408-417 ◽  
Author(s):  
Drew L. Lichtenstein ◽  
Jodi K. Craigo ◽  
Caroline Leroux ◽  
Keith E. Rushlow ◽  
R.Frank Cook ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Virus Replication ◽  
Sequence Variation ◽  
Equine Infectious Anemia Virus ◽  
Repeat Sequence ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Terminal Repeat Sequence

scholarly journals IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1602
Author(s):  
Marina Plotnikova ◽  
Alexey Lozhkov ◽  
Ekaterina Romanovskaya-Romanko ◽  
Irina Baranovskaya ◽  
Mariia Sergeeva ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Viruses ◽  
Antiviral Effect ◽  
Vero Cells ◽  
Genome Replication ◽  
Type I ◽  
Protective Effects ◽  
Cellular Models ◽  
Viral Genome Replication

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: ‘preventive’ (pretreatment); ‘preventive/therapeutic’ (pre/post); and ‘therapeutic’ (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the ‘preventive’ and ‘preventive/therapeutic’ regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

scholarly journals Characterization of the untranslated region of lymphocytic choriomeningitis virus S segment

2019 ◽  
Author(s):  
Satoshi Taniguchi ◽  
Tomoki Yoshikawa ◽  
Masayuki Shimojima ◽  
Shuetsu Fukushi ◽  
Takeshi Kurosu ◽  
...  
Keyword(s):  
Viral Genome ◽  
Lethal Dose ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Untranslated Regions ◽  
Genome Replication ◽  
Wild Type ◽  
Viral Genome Replication

ABSTRACTLymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus. The viral genome consists of two RNA segments, L and S. The 5’- and 3’-termini of both L and S segments are highly conserved among arenaviruses. These regions consist of 19 complementary base pairs and are essential for viral genome replication and transcription. In addition to these 19 nucleotides in the 5’- and 3’-termini, there are untranslated regions (UTRs) composed of 58 and 41 nucleotide residues in the 5’ and 3’ UTRs, respectively, in the LCMV S segment. Their functional roles, however, have yet to be elucidated. In this study, a reverse genetics and a minigenome system for the LCMV strain WE were established and used to analyze the function of these regions. The results obtained from these analyses, plus RNA secondary structure prediction, revealed that not only these 19 nucleotides but also the 20th–40th and 20th–38th nucleotides located downstream of the 19 nucleotides in the 5’- and 3’-termini, respectively, are heavily involved in viral genome replication and transcription. Furthermore, the introduction of mutations in these regions depressed viral propagation in vitro and enhanced attenuation in vivo. Conversely, recombinant LCMVs (rLCMVs), which had various deletions in the other UTRs, propagated as well as wild-type LCMV in vitro but were attenuated in vivo. Most mice previously infected with rLCMVs with mutated UTRs, when further infected with a lethal dose of wild-type LCMV, survived. These results suggest that rLCMVs with mutated UTRs could be candidates for an LCMV vaccine.IMPORTANCEThe function of untranslated regions (UTRs) of the arenavirus genome has not well been studied except for the 19 nucleotides of the 5’- and 3’-termini. In this study the function of the UTRs of the LCMV S segment was analyzed. It was found that not only the 19 nucleotides of the 5’- and 3’-termini but also the 20th–40th and 20th–38th nucleotides located downstream of the 19 nucleotides in the 5’- and 3’-termini, respectively, were involved in viral genome replication and transcription. Furthermore, other UTRs in the S segment were involved in virulence in vivo. The introduction of mutations to these regions makes it possible to establish attenuated LCMV and potentially develop LCMV vaccine candidates.

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