MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution.

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolved genetic interactions given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of these genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s of viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure and enables the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

Structure based sequence analysis of viral and cellular protein assemblies.

It is well accepted that, in general, protein structural similarity is strongly related to the amino acid sequence identity. To analyze in great detail the correlation, distribution and variation levels of conserved residues in the protein structure, we analyzed all available high-resolution structural data of 5245 cellular complex-forming proteins and 293 spherical virus capsid proteins (VCPs). We categorized and compare them in terms of protein structural regions. In all cases, the buried core residues are the most conserved, followed by the residues at the protein-protein interfaces. The solvent-exposed surface shows greater sequence variations. Our results provide evidence that cellular monomers and VCPs could be two extremes in the quaternary structural space, with cellular dimers and oligomers in between. Moreover, based on statistical analysis, we detected a distinct group of icosahedral virus families whose capsid proteins seem to evolve much slower than the rest of the protein complexes analyzed in this work.

VIPERdb: A Tool for Virus Research.

The VIrus Particle ExploreR database (VIPERdb) ( http://viperdb.scripps.edu ) is a database and web portal for primarily icosahedral virus capsid structures that integrates structure-derived information with visualization and analysis tools accessed through a set of web interfaces. Our aim in developing VIPERdb is to provide comprehensive structure-derived information on viruses comprising simple to detailed attributes such as size (diameter), architecture ( T number), genome type, taxonomy, intersubunit association energies, and surface-accessible residues. In addition, a number of web-based tools are provided to enable users to interact with the structures and compare and contrast structure-derived properties between different viruses. Recently, we have constructed a series of data visualizations using modern JavaScript charting libraries such as Google Charts that allow users to explore trends and gain insights based on the various data available in the database. Furthermore, we now include helical viruses and nonicosahedral capsids by implementing modified procedures for data curation and analysis. This article provides an up-to-date overview of VIPERdb, describing various data and tools that are currently available and how to use them to facilitate structure-based bioinformatics analysis of virus capsids.