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Virus structure and assembly

We study the structure and assembly of virus particles with a focus on the role of the packaged nucleic acids. The recognition, selection and packaging of viral RNA from among total cellular RNA present is critical for a viral progression. However, by sequencing the RNA encapsidated by purified Flock House Virus (FHV) particles, we have observed that FHV can package host-derived RNA transcripts including retro-transposons (Routh et al., PNAS 2012). As well as providing important insights into viral assembly and the mechanisms of RNA packaging, this revealed a potentially important role for small RNA viruses in the evolution of their hosts through the horizontal transmission of transposons. Retro-transposons were also found in virus-like-particles of FHV, which may have important implications for VLP-based therapies. We are studying the role of specific amino acids in the capsid of FHV that are known to determine faithful viral RNA packaging, as well as investigating the role of functional RNA motifs found within the RNA genome of FHV.

van de Waterbeemd M., Fort K.L., Boll D., Reinhardt-Szyba M, Routh A., Makarov A., Heck A.J.R.,
Nature Methods, 2017 Jan Early Online Access.
High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles.

Routh A., Domitrovic T., Johnson J.E.
Proceedings of the National Academy of Sciences U S A. 2012 Feb 7;109(6):1907-1912
Host RNAs, including transposons, are encapsidated by a eukaryotic ssRNA virus

Cell Cycle

Virus evolution and genetics

It has long been speculated that viruses can evolve a reduced virulence to prolong the period during which the host is infectious through the co-transmission of defective-interfering RNAs (DI-RNAs). DI-RNAs attenuate viral infections via a variety of proposed mechanisms and have been proposed to promote the transition of acute to chronic infections. Until recently, DI-RNAs had only been captured individually via classical cloning techniques, limiting our understanding of the diversity of DI-RNAs. Despite the well-established abilities of DI-RNAs to attenuate virus replication in cell culture and their observation in a number of clinical settings (e.g. measles, dengue and chronic HCV infection), little is understood about the action of DI-RNAs in live animals and their effect upon disease progression.

We are characterizing the step-wise evolution of DI-RNAs in cell culture using model systems, including Flock House Virus. In turn, we are comparing them to the DI-RNAs that arise spontaneously during live animal infections and determining their effect upon the outcome of viral infection, for instance by inducing persistence or by providing protection to super-infection. In the long-run, the ability of DI-RNAs to attenuate viral infections raises the tantalizing prospect of developing live-attenuated vaccines

Next-Generation Sequencing Technologies

We recently developed a click-chemistry based next-generation sequencing library generation method called “ClickSeq”. Here, we supplement randomly-primed RT-PCR reactions with small amounts of 3’-azido-nucleotides to randomly terminate cDNA synthesis and release a random distribution of 3’-azido blocked cDNA fragments (a process akin to classical Sanger sequencing using dideoxynucleotides). These are then ‘click-ligated’ to 5’ alkyne-modified DNA adaptors via copper-catalysed cycloaddition. This generates ssDNA molecules with unnatural yet bio-compatible triazole-linked DNA backbones that can be used as PCR templates to generate RNAseq libraries. By virtue of removing the fragmentation and enzymatic ligation steps, artifactual recombination is reduced to fewer than 3 events per million reads allowing us to confidently detect rare recombination events and replication intermediates. We are using ClickSeq to detect and profile non-homologous recombination during RNA virus passaging and the effects these events having upon virus evolution. 

In collaboration with the Wagner lab, also here in the BMB dept and UTMB, we have recently developed a method for the sequencing of Poly(A)-tail 3'UTR junctions, called Poly(A)-ClickSeq (PAC-seq). For poly(A)-ClickSeq, we initiate reverse transcription using poly(T) primers, without a non-T anchor. In this manner, we can specifically reverse transcribe the 3’ ends of total cellular RNA directly from crude RNA extracts. However, the critical innovation used is to omit AzTTP from the reaction mixture (i.e. we provide a mixture of AzVTPs and dNTPs). This means that reverse-transcription cannot terminate opposite an ‘A’ in the RNA template. Rather, reverse-transcription must continue until non-A residues are found, essentially ‘homing-in’ on the junction of the 3’UTR and the poly(A) tail. cDNA synthesis is stochastically terminated at a distance upstream of the 3’UTR/poly(A) junction tailored by the AzVTP:dNTP ratio. Therefore, we can purify the azido-terminated cDNA, click-ligate our 5’ Illumina adaptor and generate an NGS library enriched with 3’UTR/poly(A) junctions

Routh A.*, Head S.R., Ordoukhanian P., Johnson J.E.
Journal of Molecular Biology, 2015 Aug 14;427(16):2160-6
ClickSeq: Fragmentation-free next-generation sequencing via click-ligation of adaptors to stochastically terminated 3’-azido cDNAs.

Routh A.*, Ji P., Jaworski E., Xia Z., Li W. and Wagner E.J.*
Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation.

Poly(A)-ClickSeq for poly(A) tail sequencing Figure_RNA2_DIs

Computational Virology:

We are utilizing and developing computational pipelines for the analysis of NGS data of viral samples.

ViReMa” (Viral Recombination Mapper) is a versatile and flexible computational pipeline for the discovery of viral recombination events in NGS datasets that employs a novel ‘moving-seed’ approach for sequence alignment (Routh et al 2014 NAR). In addition to improved speed and sensitivity over other algorithms using canonical ‘fixed-seed’ approaches, ViReMa detects substitutions and non-reference insertions, multiple recombination events and virus to host recombination. This flexibility has proven critical for mapping viral recombinations as these events rarely conform to predefined (or known) rules. Using ViReMa, we have found that after resistance to protease inhibitors had developed in HIV positive patients, virus populations haboured short duplications proximal to the proteolysis sites in the GAG protein. Sourceforge-link

Routh A.*, Johnson J.E.
Nucleic Acids Research, 2014 Jan 42(2):e11
Discovery of functional genomic motif in viruses with ViReMa – a Virus Recombination Mapper for analysis of Next-Generation Sequencing data

“CoVaMa” (Co-Variation Mapper) takes NGS alignment data and populates large matrices of contingency tables that correspond to every possible pairwise interaction of nucleotides in the viral genome or amino acids in the chosen open reading frame (Routh et al. 2015 Methods). These tables are then analysed using classical linkage disequilibrium to detect and report evidence of epistasis. CoVaMa found epistatically linked loci in FHV genomic RNA grown under controlled cell culture conditions as well as correlated amino acid substitutions in the protease genes among a large cohort of HIV infected patients undergoing anti-retroviral therapy. Sourceforge-link

Routh A.*, Chang M.W., Okulicz J.F., Johnson J.E., Torbett B.E.*
Methods, 2015 Dec 91:40-47
CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data.