Hockman, M. R. | Search bioRxiv

bioRxiv2019

A method for the unbiased quantification of reassortment in segmented viruses

The diversification of segmented viruses via reassortment is important to understand due to the contributions of reassortment to viral evolution and emergence. Methods for the quantification of reassortment have been described, but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of unbiased reassortment and can be applied to diverse segmented viruses.\n\nHighlightsO_LIGenetic tagging of viruses can be achieved without altering fitness\nC_LIO_LIHigh-resolution melt can detect single nucleotide differences in viruses\nC_LIO_LIUnbiased reassortment of influenza A virus and mammalian orthoreovirus can be quantified\nC_LI

microbiology↗

bioRxiv2019

Dysregulation of M segment gene expression contributes to influenza A virus host restriction

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.\n\nAuthor summaryInfluenza A virus (IAV) pandemics arise when a virus adapted to a non-human host overcomes species barriers to successfully infect humans and sustain human-to-human transmission. To gauge the adaptive potential and therefore pandemic risk posed by a particular IAV, it is critical to understand the mechanisms underlying viral adaptation to human hosts. Here, we focused on the role of one of IAVs eight gene segments, the M segment, in host adaptation. Comparing the growth of IAVs with avian- and human-derived M segments in avian and mammalian systems revealed that the avian M segment restricts viral growth specifically in mammalian cells. We show that the mechanism underlying this host range phenotype is a dysregulation of viral gene expression when the avian IAV M segment is transcribed in mammalian cells. In particular, excess production of the M2 protein results in viral interference with cellular functions on which the virus relies. Our results therefore reveal that the use of cellular machinery to control viral gene expression leaves the virus vulnerable to over- or under-production of critical viral gene products in a new host species.

microbiology↗