Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature

Abstract

Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world’s most prevalent pathogens and could aid target selection for vaccine or antiviral development.

Contemporary Enterovirus D68 strains show enhanced replication and translation at 37°C

“…EV-D68 has previously been shown to have an optimal growth temperature of 32°C compared to 37°C [1,4,5]. This temperature range is important because it represents the temperature of the human upper respiratory airways (32°C), which are cooled by inhalation of ambient air, and the lower respiratory airways (37°C), closer to core body temperature.…”

“…Our results suggest that determinants outside the 5' UTR control temperature depending infectious virus production. Recent literature suggests that EV-D68 temperature sensitivity may be linked to the viral capsid protein VP1 [4]. Specifically, by taking a temperature tolerant EV-D94 virus and swapping the VP1 with that of the prototype EV-D68 strain (Fermon-1962), the virus becomes temperature sensitive.…”

“…Historic strains of EV-D68 were previously shown to have optimal growth temperatures at 33°C and undergo attenuated growth at 37°C [1,4,5]. Given the recent rise in acute respiratory illness correlating with EV-D68, we tested a panel of historic and contemporary strains to determine if temperature sensitivity phenotypes differ between historic and contemporary strains.…”

“…This positive-sense, single-stranded RNA virus of the Picornaviridae family was first isolated in California from four pediatric patients in 1962. EV-D68 was found to be remarkably similar to human rhinovirus (HRV) in that it was acid labile, temperature sensitive, and transmitted from person-to-person via respiratory droplets [1][2][3][4][5]. The biological similarities between in EV-D68 and HRV led to its initial misclassification as human rhinovirus 87, which was corrected by genetic analysis and serum neutralization studies [6,7].…”

“…It is widely accepted that the reason most HRVs cause only mild upper respiratory illness is their preferential replication at cooler physiological temperatures [52][53][54].Temperature sensitivity and restriction to the upper airways has been harnessed for vaccine development and is the basis behind the cold-adapted live attenuated influenza vaccine [55,56]. Historically, in vitro studies indicated EV-D68 replicates optimally at 33°C and is attenuated at higher physiological temperatures [1,4,5].…”