Research Article: Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

Date Published: March 13, 2014

Publisher: Public Library of Science

Author(s): Greg Brennan, Jacob O. Kitzman, Stefan Rothenburg, Jay Shendure, Adam P. Geballe, Richard C. Condit.


The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.

Partial Text

There are at least 868 described zoonotic microbial pathogens, 33% of which are capable of human to human transmission [1]. Recent viral zoonoses have led to some of the most devastating and medically relevant outbreaks in modern history, including SARS coronavirus, pandemic influenza, and HIV/AIDS, highlighting the urgent need to understand how viruses adapt to infect new species. At a population level, factors influencing the transmission of zoonotic pathogens to humans include increasing population density, greater contact with wildlife, increased travel, and poor public health infrastructure [2], [3]. However, these factors only allow the microbe increased access to new hosts; they do not directly enable it to adapt to and replicate in the new species. Intermediate hosts, animals that are not the natural host of a virus but are still permissive or semi-permissive for viral replication, play a critical role in cross-species transmission. These hosts can facilitate increased contact between a virus and a new host, and drive adaptive changes that may improve virus replication (Reviewed in [4]). For example, spill-over of Nipah virus from fruit bats into pigs, the intermediate host, increased human exposure to the virus and resulted in eventual human outbreaks in Malaysia [5], [6]. In another example, lentiviral adaptation through intermediate chimpanzee hosts led to both increased contact with humans, and adaptive genetic changes permitting the virus to inhibit the human versions of several host restriction factors (Reviewed in [7]).

Cross-species pathogen transmissions have been responsible for more than 60% of all emerging infectious diseases in humans during the past 70 years [24]. Human immunodeficiency viruses, avian influenza viruses, and the recently described Middle East respiratory syndrome coronavirus exemplify the ongoing threat and potential of animal viruses to spread to and among humans, highlighting the urgent need to understand the mechanisms underlying cross-species transmission and adaptation to new hosts. One such mechanism, genetic locus amplification in response to selective pressure, has been observed in both viruses and bacteria [16]–[18], [21]. Here we demonstrate that amplification of the exogenous gene rhtrs1 is sufficient to block potent PKR-mediated inhibition and improve VVΔEΔK+RhTRS1 replication in AGM-derived PRO1190 cells. This adaptation also expanded the species tropism of the virus, enabling markedly improved replication in otherwise resistant human and rhesus monkey cells. Importantly, VVΔEΔK+RhTRS1 failed to replicate in HFF to a level sufficient to sustain transmission upon serial passage, demonstrating that adaptation in PRO1190 was a critical intermediate step to expand the viral species tropism. Thus, the process of adaptation in one host may increase the likelihood of virus transmission to a variety of divergent species.




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