Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha’s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Hysteretic hERG Channel Gating Current Recorded At Physiological Temperature

Cardiac hERG channels comprise at least two subunits, hERG 1a and hERG 1b, and drive cardiac action potential repolarization. hERG 1a subunits contain a cytoplasmic PAS domain that is absent in hERG 1b. The hERG 1a PAS domain regulates voltage sensor domain (VSD) movement, but hERG VSD behavior and its regulation by the hERG 1a PAS domain have not been studied at physiological temperatures. We recorded gating charge from homomeric hERG 1a and heteromeric hERG 1a/1b channels at near physiological temperatures (36 ± 1°C) using pulse durations comparable in length to the human ventricular action potential. The voltage dependence of deactivation was hyperpolarized relative to activation, reflecting VSD relaxation at positive potentials. These data suggest that relaxation (hysteresis) works to delay pore closure during repolarization. Interestingly, hERG 1a VSD deactivation displayed a double Boltzmann distribution, but hERG 1a/1b deactivation displayed a single Boltzmann. Disabling the hERG1a PAS domain using a PAS-targeting antibody similarly transformed hERG 1a deactivation from a double to a single Boltzmann, highlighting the contribution of the PAS in regulating VSD movement. These data represent, to our knowledge, the first recordings of hERG gating charge at physiological temperature and demonstrate that VSD relaxation (hysteresis) is present in hERG channels at physiological temperature.

1D-Zigzag Eu3+/Tb3+ Coordination Chains as Luminescent Ratiometric Thermometers Endowed with Multicolor Emission

Two homometallic Coordination Polymers (CPs) with composition [Ln(hfac)3bipy]n (Ln3+ = Eu3+, 1, and Tb3+, 2; hfac = hexafluoroacetylacetonato, bipy = 4,4′-bipyridine) were used to develop a family of ratiometric luminescent thermometers containing Eu3+ and Tb3+ as red and green emitters, respectively. The thermometric properties of pure CPs and of their mixtures having an Eu3+/Tb3+ molar ratio of 1:1, 1:3, 1:5, and 1:10 (samples: Eu1Tb1, Eu1Tb3, Eu1Tb5, and Eu1Tb10) were studied in the 83–383 K temperature range. Irrespective of the chemical composition, we observed similar thermometric responses characterized by broad applicative temperature ranges (from 100 to 165 K wide), and high relative thermal sensitivity values (Sr), up to 2.40% K−1, in the physiological temperature range (298–318 K). All samples showed emissions endowed with peculiar and continuous color variation from green (83 K) to red (383 K) that can be exploited to develop a colorimetric temperature indicator. At fixed temperature, the color of the emitted light can be tuned by varying composition and excitation wavelength.

Transsynaptic N-Cadherin Adhesion Complexes Control Presynaptic Vesicle and Bulk Endocytosis at Physiological Temperature

At mammalian glutamatergic synapses, most basic elements of synaptic transmission have been shown to be modulated by specific transsynaptic adhesion complexes. However, although crucial for synapse homeostasis, a physiological regulation of synaptic vesicle endocytosis by adhesion molecules has not been firmly established. The homophilic adhesion protein N-cadherin is localized at the peri-active zone, where the highly temperature-dependent endocytosis of vesicles occurs. Here, we demonstrate an important modulatory role of N-cadherin in endocytosis at near physiological temperature by synaptophysin-pHluorin imaging. Different modes of endocytosis including bulk endocytosis were dependent on N-cadherin expression and function. N-cadherin modulation might be mediated by actin filaments because actin polymerization ameliorated the knockout-induced endocytosis defect. Using super-resolution imaging, we found strong recruitment of N-cadherin to glutamatergic synapses upon massive vesicle release, which might in turn enhance vesicle endocytosis. This provides a novel, adhesion protein-mediated mechanism for efficient coupling of exo- and endocytosis.

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures

Chikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitoes and causing fever, rash and arthralgia. Currently there are no vaccines or antiviral agents against CHIKV, therefore it is important to understand the molecular details of CHIKV replication. In this regard, the function of the Alphavirus non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. We identified three mutants that replicated poorly in mammalian cells but showed no defect in mosquito cells. We further showed that these mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28°C). Similar effects were observed in the context of infectious CHIKV as well as a closely related virus: O’Nyong Nyong virus (ONNV). Intriguingly, this analysis also revealed that the wildtype SGR replicated much more efficiently at sub-physiological temperature as compared to 37°C. This was not due to impaired interferon responses as this enhancement was also observed in Vero cells. Neither was this due to a defect in the phosphorylation of eIF2α as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37°C. However, we noticed significant differences between the sizes and numbers of virus-induced stress granules (SG) at physiological and sub-physiological temperatures. As cells in the periphery will be at sub-physiological temperatures, and these will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses have evolved mechanisms to limit antiviral responses in these cells to promote viral genome replication.Author summaryChikungunya virus (CHIKV) is a re-emerging arbovirus. It is transmitted by Aedes species of mosquitos and poses massive epidemic threats. Arboviruses are unique in that they must be able to replicate efficiently in both the mosquito vector and the mammalian host, and therefore at different temperatures. Importantly, the first cells infected in the mammalian host following a mosquito bite will be in the skin and therefore at sub-physiological temperature (lower than 37°C). Here we show that mutants within one of the CHIKV proteins (nsP3) were unable to replicate at 37°C but replicated efficiently in mammalian cells at a sub-physiological temperature (28°C). We also showed that the wildtype virus replicated more efficiently at 28°C in comparison to at 37°C in mammalian cells. We investigated the mechanism behind this phenomenon and showed that at sub-physiological temperatures the virus induced the formation of smaller and more numerous cytoplasmic stress granules. We propose that alphaviruses have evolved mechanisms to promote their replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a cutaneous mosquito bite.