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.

Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions.

BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters. The most common type of cooperativity is supported by direct protein-protein interactions. Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro. probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site. This mechanism does not require that the transcription factors have activation domains. We have examined whether this phenomenon occurs in vivo. RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances. In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains. CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates. These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo.

Most proteins that activate RNA polymerase II-mediated transcription in eukaryotic cells contain sequence-specific DNA-binding domains and "activation" regions. The latter bind general transcription factors and/or coactivators and are required for high-level transcription. Their function in vivo is unknown. Since several activation domains bind the TATA-binding protein (TBP), TBP-associated factors, or other general factors in vitro, one role of the activation domain may be to facilitate promoter occupancy by supporting cooperative binding of the activator and general transcription factors. Using the GAL4 system of yeast, we have tested this model in vivo. It is demonstrated that the presence of a TATA box (the TBP binding site) facilitates binding of GAL4 protein to low- and moderate-affinity sites and that the activation domain modulates these effects. These results support the cooperative binding model for activation domain function in vivo.

Synergistic activation of transcription by physiologically unrelated transcription factors through cooperative DNA-binding.

Most eukaryotic promoters contain binding sites for several different transcription factors, which often act synergistically. Mechanistically, synergy is ascribed either to cooperative DNA-binding of the factors to the promoter or to some type of "multiple contact" mechanism in which each activator performs a different task in stimulating the transcription machinery. Here, it is shown that the yeast activators Gal4 and Put3 bind to DNA cooperatively in vivo and can activate transcription synergistically from certain synthetic promoters. Normally, Gal4 and Put3 bind to completely different promoters and activate physiologically unrelated sets of genes and it is extremely unlikely that they have evolved direct protein-protein contacts. These studies add to a growing body of evidence that binding of proteins to nearby sites in chromatin is intrinsically cooperative and suggest that many examples of synergy ascribed to multiple contact mechanisms may instead involve non-traditional cooperative DNA-binding.

How do "Zn2 cys6" proteins distinguish between similar upstream activation sites? Comparison of the DNA-binding specificity of the GAL4 protein in vitro and in vivo.

The GAL4 protein of Saccharomyces cerevisiae is the prototype of a family of transcription factors that contain a "Zn2Cys6" coordination complex in the DNA-binding domain. GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sites that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3'. Other Zn2Cys6 proteins in S. cerevisiae also recognize sequences containing two CGG triplets, but with different spacings between them. In this report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk genomic DNA. In vitro, GAL4 recognizes with moderate to high affinity a variety of sites of the general formula (A/C)GGN10-12CCG. This level of specificity is apparently insufficient for the activator to carry out its biological role. However, many of the sites to which GAL4 binds in vitro do not support GAL4-activated transcription in vivo. In most cases there is not a quantitative correlation between the relative affinity of a site for GAL4 in vitro and the level of GAL4-dependent transcription supported by it in vivo. These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 is modified.

Assembly of the human origin recognition complex.

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.