Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

Solórzano, Alicia; Webby, Richard J.; Lager, Kelly M.; Janke, Bruce H.; Garcı́a-Sastre, Adolfo; Richt, Jürgen A.

doi:10.1128/jvi.79.12.7535-7543.2005

Cited by 221 publications

(272 citation statements)

References 58 publications

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“…In pigs vaccinated with the attenuated NS1 126 TX98 mutant, the mean viral titers did not exceed 10 TCID 50 /ml on days 2 or 4, and no virus was detected on day 6. This disparity in wild type TX98 and NS1 126 TX98 replication in vivo is consistent with the attenuated phenotype in previously reported results [17].…”

Section: Vaccine Virus Replication In Naïve Pigssupporting

confidence: 92%

“…However, given the very low level of NS1 126 TX98 virus detected in pigs' nasal swabs after intranasal inoculation, it is also possible that the vaccine did not supply enough endogenous antigen to prime a large population of CD4 -CD8 + T cells. Viruses engineered with even shorter NS1 proteins than that of NS1 126 TX98 are paradoxically less attenuated, in both pigs and mice [17,30]. It is reasonable to predict that mutants such as those would elicit greater CD4 -CD8 + T cell responses.…”

Section: Discussionmentioning

confidence: 99%

“…The same strains were grown in MDCK cell cultures to generate recall antigen for ex vivo stimulation of T cells. Live influenza A virus NS1 126 TX98 was generated by reverse genetics, as previously described [17], and propagated in allantoic cavities of 10-day old embryonated chicken eggs to produce an inoculum for vaccination. Sequences of IA04 were generated by 454 genome sequencing technology, complemented by Sanger sequencing to fill gaps, as described previously [24].…”

Section: Virusesmentioning

confidence: 99%

“…Molecular approaches have been used to construct mutated SIV genomes which confer attenuated replication properties, including reduced NS1 suppression of type I interferon, dependence on the enzyme elastase for HA cleavage, and temperature-sensitive mutations in polymerase genes [17][18][19]. Truncation of NS1 protein in triple reassortant H3N2 strain A/Sw/Texas/4199-2/98, from a length of 230 amino acids to 126, produced a mutant with restricted replication in the swine respiratory tract but strong immunogenic properties [20].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

Kappes

Sandbulte

Platt

et al. 2012

Vaccine

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The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad crossprotection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4 + CD8 -, CD4 + CD8 + , CD4 -CD8 + , and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4 + CD8 + cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains. The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1 126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1 126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1 126 TX98 infected pigs were minimal. However, intracellular IFN-␥ data indicate that the attenuated virus induced virus-specific CD4 + CD8 -, CD4 + CD8 + , CD4 -CD8 + , and ␥␦ T cells within 28 days. The IFN-␥ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4 + CD8 + cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replicatio...

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Section: Vaccine Virus Replication In Naïve Pigssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Virusesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

Kappes

Sandbulte

Platt

et al. 2012

Vaccine

Self Cite

View full text Add to dashboard Cite

show abstract

“…Interferon inhibition assays were performed according to the general methodology established by Rehwinkel et al to trigger IFN production using an active influenza virus RNA-dependent RNA polymerase (vRdRp) (44) and a bioassay established by Solorzano et al (45), using the VSV-GFP developed by Stojdl et al (46). Briefly, HEK293FT cells were transfected with various combinations of plasmids, including those needed to produce a functional vRdRp [here referred to as PolII(PB2ϩPB1ϩPAϩNP)] and a construct transcribing the PB2 gene segment under an RNA polymerase I promoter in the reverse complementary direction [here referred to as PolI(PB2)].…”

Section: Methodsmentioning

confidence: 99%

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

Santos

Pal

Chacon

et al. 2013

J Virol

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Our pioneering studies on the interplay between the small ubiquitin-like modifier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ϳ4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.

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Using Reverse Genetics to Improve Influenza Vaccines

Elderfield

Hartgroves

Barclay

2012

Reverse Genetics of RNA Viruses

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Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

Cited by 221 publications

References 58 publications

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

Using Reverse Genetics to Improve Influenza Vaccines

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