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Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

ABSTRACT: Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3D56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3D56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

SUBMITTER: Patterson EI

PROVIDER: S-EPMC7291308 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

Patterson Edward I EI Khanipov Kamil K Swetnam Daniele M DM Walsdorf Samantha S Kautz Tiffany F TF Thangamani Saravanan S Fofanov Yuriy Y Forrester Naomi L NL

Viruses 20200515 5

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3D56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine stra ...[more]

PMID: 32429270

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Project description:Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide.

Project description:BackgroundMembers of the phylum Chlamydiae are obligate intracellular pathogens of humans and animals and have a serious impact on host health. They comprise several zoonotic species with varying disease outcomes and prevalence. To investigate differences in virulence, we focused on Chlamydia psittaci, C. abortus and Waddlia chondrophila. Most threatening is C. psittaci, which frequently infects humans and causes psittacosis associated with severe pneumonia. The closest relative of C. psittaci is C. abortus, which shares the vast majority of genes but less frequently infects humans, and causes stillbirth and sepsis. W. chondrophila is more distantly related, and occasional human infections are associated with respiratory diseases or miscarriage. One possible explanation for differences in virulence originate from species-specific genes as well as differentially expressed homologous virulence factors.ResultsRNA-sequencing (RNA-Seq) was applied to purified infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs) in order to elucidate the transcriptome of the infectious and replicative chlamydial states. The results showed that approximately half of all genes were differentially expressed. For a descriptive comparison, genes were categorised according to their function in the RAST database. This list was extended by the inclusion of inclusion membrane proteins, outer membrane proteins, polymorphic membrane proteins and type III secretion system effectors. In addition, the expression of fifty-six known and a variety of predicted virulence and immunogenic factors with homologs in C. psittaci, C. abortus and W. chondrophila was analysed. To confirm the RNA-Seq results, the expression of nine factors was validated using real-time quantitative polymerase chain reaction (RT-qPCR). Comparison of RNA-Seq and RT-qPCR results showed a high mean Pearson correlation coefficient of 0.95.ConclusionsIt was shown that both the replicative and infectious chlamydial state contained distinctive transcriptomes and the cellular processes emphasised in EBs and RBs differed substantially based on the chlamydial species. In addition, the very first interspecies transcriptome comparison is presented here, and the considerable differences in expression of homologous virulence factors might contribute to the differing infection rates and disease outcomes of the pathogens. The RNA-Seq results were confirmed by RT-qPCR and demonstrate the feasibility of interspecies transcriptome comparisons in chlamydia.

Dataset Information

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

Publications

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

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