Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.

The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in ϳ15-to 50-fold more efficient establishment of persistent replication in hamster ( (29), with one long open reading frame coding for 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (7). The gene order of KUN genome RNA is 5Ј-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3Ј. We previously constructed the first flavivirus replicons based on KUN cDNA by deleting the majority of the genomic region including structural genes (30) and used them extensively for the development of a gene expression system (2,19,28,30,44,45). KUN replicons were also used extensively in RNA replication and complementation studies (23)(24)(25)(26)(27)32) that contributed substantially to generating a comprehensive model for the formation and operation of the flavivirus RNA replication complex (47,48).The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses was shown to lead to the generation of adaptive mutations in nonstructural genes that either decreased (alphavirus replicons) or enhanced (hepatitis C replicons) RNA replication efficiency (1,4,13,14,34,39,41,50). Some of these adaptive mutations (e.g., in the NS5A protein of the hepatitis C replicon) were shown to increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤) (41), while others (e.g., in the nsP2 protein of Sindbis virus) were shown to lead to more efficient IFN production (14). IFN response is the first line of cell defense against viral infections, and a majority of viruses have developed various strategies to overcome it, by either inhibiting IFN production or blocking IFN signaling (15,22,35,43). Recent studies of dengue virus have indicated that flaviviruses may interfere with early steps of IFN signaling and have implied roles for the small nonstructural proteins NS2A, NS4A, and NS4B in this process (37).In this study, we describe the identification of adaptive mutations in KUN replicon RNA that confirm an advantage in establishing persistent replication in a number of different cell lines. We also demonstrate that induction of IFN-␤ promoter-* Correspondi...

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“…per ml. Vero cells are known to have a deletion of the IFN-␤ gene (36), and BHK21 cells also appear to have a defect in IFN production (38).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription

Liu

Chen

Wang

et al. 2004

J Virol

152

126

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“…In addition, such IFNsensitive viruses may be difficult to grow in culture, because most tissue-culture cells can produce and respond to IFN following infection. Vero cells can be used, as they do not produce IFN due to natural deletion of the IFN genes (Desmyter et al, 1968;Mosca & Pitha, 1986). They have the additional advantage that they are already used in manufacture of virus vaccines.…”

Section: Ifn-sensitive Viruses As Attenuated Virus Vaccinesmentioning

confidence: 99%

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

Randall

Goodbourn

2008

Journal of General Virology

1,394

1,420

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The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most, if not all, virus infections in the absence of adaptive immunity. However, viruses can still replicate and cause disease in vivo, because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn, S., Didcock, L. & Randall, R. E. (2000). J Gen Virol 81, 2341-2364] but, since then, a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest, but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review, we describe how viruses activate the IFN system, how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part, we describe how viruses circumvent the IFN response. Here, we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections), whether perturbation of the IFN response by persistent infections can lead to chronic disease, and the importance of the IFN system as a species barrier to virus infections. Lastly, we briefly describe applied aspects that arise from an increase in our knowledge in this area, including vaccine design and manufacture, the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer. Biology of the interferon systemThe interferons (IFNs) are a group of secreted cytokines that elicit distinct antiviral effects. They are grouped into three classes called type I, II and III IFNs, according to their amino acid sequence. Type I IFNs (discovered in 1957;Isaacs & Lindenmann, 1957) comprise a large group of molecules; mammals have multiple distinct IFN-a genes (13 in man), one to three IFN-b genes (one in man) and other genes, such as IFN-v, -e, -t, -d and -k. The IFN-a and -b genes are induced directly in response to viral infection, whereas IFN-v, -e, -d and -k play less well-defined roles, such as regulators of maternal recognition in pregnancy. Thus, rather than use the term 'type I IFN', we will use IFN-a/b when referring to the virally induced cytokines. Although the multigenic nature of IFN-a has been known for over 20 years, the significance of this is still debatedi.e. whether these genes are expressed differentially in distinct cell types, whether they are inducible by different types of viruses or whether they are functionally specialized (Brideau-Andersen et al., 2007). For the rest of this review, we will not distinguish between IFN-a subtypes. Type III IFNs have been described more recently and comprise IFNl1, -l2 and -l3, also referred to as IL-2...

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“…By contrast, the wild-type virus encodes an NS1 protein of 230 amino acids. We infected Vero cells, a cell line that is known to lack the type I IFN loci (17,18), with NS1-99, delNS1, or PR8 virus at an MOI of 2. Despite the alterations in the NS gene, all three of these influenza A viruses express comparable levels of the viral NEP (NS2), the second protein encoded by the viral NS gene ( Fig.…”

Section: Growth Of Ns1-altered Influenza a Viruses In Embryonated Eggsmentioning

confidence: 99%

Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach

Talon

Salvatore

O’Neill

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

291

274

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We propose a rational approach to the generation of live viral vaccines: alteration of virally encoded type I IFN antagonists to attenuate virulence while retaining immunogenicity. We have explored this concept by using the influenza virus. Previously we have shown that the NS1 protein of influenza A virus possesses anti-IFN activity. We now present evidence that influenza A and B viruses encoding altered viral NS1 proteins are highly attenuated in the mouse host, yet provide protection from challenge with wild-type viruses.

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Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.

Abstract: We determined that the defect in beta interferon induction in Vero cells is due to the absence of the simian beta interferon (IFN-j)

Cited by 186 publications

References 22 publications

Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription

Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach

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