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A novel self-replicating chimeric lentivirus-like particle.

ABSTRACT: Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4? cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

SUBMITTER: Jurgens CK

PROVIDER: S-EPMC3255904 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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A novel self-replicating chimeric lentivirus-like particle.

Jurgens Christy K CK Young Kelly R KR Madden Victoria J VJ Johnson Philip R PR Johnston Robert E RE

Journal of virology 20111019 1

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vac ...[more]

PMID: 22013035

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Project description:BackgroundThe HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.ResultsBy co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV(MAC239)Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.ConclusionsWe have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.

Project description:Numerous varieties of life forms have filled the earth throughout evolution. Evolution consists of two processes: self-replication and interaction with the physical environment and other living things around it. Initiated by von Neumann et al. studies on self-replication in cellular automata have attracted much attention, which aim to explore the logical mechanism underlying the replication of living things. In nature, competition is a common and spontaneous resource to drive self-replications, whereas most cellular-automaton-based models merely focus on some self-protection mechanisms that may deprive the rights of other artificial life (loops) to live. Especially, Huang et al. designed a self-adaptive, self-replicating model using a greedy selection mechanism, which can increase the ability of loops to survive through an occasionally abandoning part of their own structural information, for the sake of adapting to the restricted environment. Though this passive adaptation can improve diversity, it is always limited by the loop's original structure and is unable to evolve or mutate new genes in a way that is consistent with the adaptive evolution of natural life. Furthermore, it is essential to implement more complex self-adaptive evolutionary mechanisms not at the cost of increasing the complexity of cellular automata. To this end, this article proposes new self-adaptive mechanisms, which can change the information of structural genes and actively adapt to the environment when the arm of a self-replicating loop encounters obstacles, thereby increasing the chance of replication. Meanwhile, our mechanisms can also actively add a proper orientation to the current construction arm for the sake of breaking through the deadlock situation. Our new mechanisms enable active self-adaptations in comparison with the passive mechanism in the work of Huang et al. which is achieved by including a few rules without increasing the number of cell states as compared to the latter. Experiments demonstrate that this active self-adaptability can bring more diversity than the previous mechanism, whereby it may facilitate the emergence of various levels in self-replicating structures.

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A novel self-replicating chimeric lentivirus-like particle.

Publications

A novel self-replicating chimeric lentivirus-like particle.

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