Temperature-sensitive mutants of influenza A virus. Transfer of the twots-1 A 2ts lesions present in the Udorn/72-ts-1 A 2 donor virus to the influenza A/Alaska/6/77 (H 3 N 2) wild type virus

1980 ◽  
Vol 65 (2) ◽  
pp. 175-186 ◽  
Author(s):  
B. R. Murphy ◽  
F. T. Wood ◽  
J. G. Massicot ◽  
R. M. Chanock
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Type Virus ◽  
Wild Type ◽  
Temperature Sensitive Mutants ◽  
Virus Transfer

Temperature-Sensitive Mutants of Influenza A Virus: Transfer of the Two Temperature-Sensitive Lesions in the Udorn/72- ts -1A2 Virus to the A/Hong Kong/123/77 (H1N1) Wild-Type Virus

1980 ◽  
Vol 28 (3) ◽  
pp. 792-798
Author(s):  
Brian R. Murphy ◽  
Nanette T. Hosier ◽  
Robert M. Chanock
Keyword(s):  
Hong Kong ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Genetic Interaction ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Virus Transfer

The influenza A/Udorn/72- ts -1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37°C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts -1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77- ts -1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts -1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77- ts -1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

2016 ◽  
Vol 90 (7) ◽  
pp. 3684-3693 ◽  
Author(s):  
Léa Meyer ◽  
Alix Sausset ◽  
Laura Sedano ◽  
Bruno Da Costa ◽  
Ronan Le Goffic ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

scholarly journals The Morphology and Composition of Influenza A Virus Particles Are Not Affected by Low Levels of M1 and M2 Proteins in Infected Cells

2005 ◽  
Vol 79 (12) ◽  
pp. 7926-7932 ◽  
Author(s):  
Svetlana V. Bourmakina ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles ◽  
M1 Protein ◽  
Infected Cells ◽  
Low Levels ◽  
Viral Components

ABSTRACT We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.

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