SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis.

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. Significance: A hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.

RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.

In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level.

Genetic differences in the time course for the development and persistence of the anticonvulsant effects of carbamazepine against cocaine seizures.

Initial studies of the effect of chronic carbamazepine (CBZ) against cocaine-induced seizures indicated that there were genetic differences in both the time course for the development of the anticonvulsant effects of CBZ against cocaine-induced seizures and the persistence of these effects. The present studies were initiated to investigate the time course for the development and persistence of the anticonvulsant effects of chronic CBZ against cocaine seizures in BALB/cByJ, C57Bl/6J and SJL/J mice. The anticonvulsant actions of CBZ were dependent on the duration of CBZ administration, requiring 4-7 days to achieve maximal efficacy. However, once the anticonvulsant effects of CBZ were manifest, the effect persisted for up to 5 days after stopping CBZ treatment depending on the genotype. The levels of CBZ and its active epoxide metabolite were determined in plasma and brain at various time points during and after chronic CBZ treatment. The levels of CBZ and CBZ-10,11-epoxide were substantially reduced over the course of treatment in all three strains, such that the levels of the two compounds in plasma and brain could not account for the decreased susceptibility to cocaine seizures observed following chronic CBZ. These results suggest that the effects of CBZ on cocaine seizures are mediated by relatively long-term changes in one or more biological systems associated with cocaine's convulsant effects.

REM need in adolescents as indicated by resistance to REM deprivation.

The hypothesis that adolescents have an exceptionally strong need for REM sleep was tested by measuring their resistance to REM deprivation. Ten adolescents (aged 16 to 17 yr.) were compared with 12 young adults (aged 25 to 27 yr.) in a standard REM deprivation procedure. The adolescents had to be awakened significantly more times than the young adults; this is consistent with the hypothesis that adolescents have a greater need for REM sleep.