Dataset Information

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

ABSTRACT: Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.

SUBMITTER: Ziegler CM

PROVIDER: S-EPMC4806877 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Publications

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

Ziegler Christopher M CM Eisenhauer Philip P Bruce Emily A EA Weir Marion E ME King Benjamin R BR Klaus Joseph P JP Krementsov Dimitry N DN Shirley David J DJ Ballif Bryan A BA Botten Jason J

PLoS pathogens 20160324 3

Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein ...[more]

PMID: 27010636

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Project description:Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. KEY POINTS: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers.

Dataset Information

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

Publications

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

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