A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants.

Next-generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the world; however, it is costly and time-consuming to perform and is not widely available in low-resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)-PCR-pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS-CoV-2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS-CoV-2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Evaluation of transport media for laboratory detection of SARS-CoV-2 in upper respiratory tract swab specimens.

The reduced availability of commercial swabs and transport media for testing and administrative demands for increased testing capacity during the coronavirus disease 2019 (COVID-19) public health emergency has seriously challenged national laboratory testing programs, forcing many to use nontraditional collection devices, often without typical analytical assessment of their suitability in testing. Five common transport media (four commercial and one in-house) were evaluated for their suitability in the collection of nasopharyngeal swab specimens for subsequent molecular detection of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). Results suggest that these transport media provide dependable temporal stability of the SARS-CoV-2 virus without significant analytical interference of molecular assays. These findings are not only important for addressing critical laboratory supply chain shortages of transport media in the current COVID-19 health crisis but also for future pandemic planning, when again supplies of commercially available transport media might be depleted.

Regulation of the association of the PAF53/PAF49 heterodimer with RNA polymerase I.

Mammalian PAF49 and PAF53 form a heterodimer and are essential for transcription. However their roles in transcription have not been specifically defined. While the yeast homologues are "not essential" proteins, yeast cells deficient in the homologue of PAF53 grow at 50-66% the wild-type rate at 30°C, but fail to grow at 25°C (Liljelund et al., 1992; Beckouet et al., 2008). There is increasing evidence that these proteins may play important roles in transcription initiation and elongation. We have found that while some cells regulated the protein levels of both PAF53 and PAF49, other cells did not. However, in either case they regulated the nucleolar levels of the PAFs. In addition, we found that the association of PAF49/PAF53 with Pol I is regulated. In examining the mechanism that might regulate this association, we have found that PAF49 is acetylated on multiple sites. The acetylation state of PAF49 does not affect heterodimerization. However, hypoacetylated heterodimer binds to Pol I with greater affinity than acetylated heterodimer. Further, we have found that the heterodimer interacts with Rrn3. We propose a model, in which there is a biochemical interaction between the Pol I-associated heterodimer and Rrn3 and that this interaction facilitates the recruitment of Rrn3 to the polymerase. As the binding of Rrn3 to Pol I is essential to transcription initiation in yeast and mammals, our results provide a greater understanding of the regulation of Rrn3 function and provide biochemical underpinning for the roles of the PAF49/PAF53 heterodimer in transcription initiation and elongation by Pol I.

Selective inhibition of rDNA transcription by a small-molecule peptide that targets the interface between RNA polymerase I and Rrn3.

UNLABELLED: The interface between the polymerase I-associated factor Rrn3 and the 43-kDa subunit of RNA polymerase I is essential to the recruitment of Pol I to the preinitiation complex on the rDNA promoter. In silico analysis identified an evolutionarily conserved 22 amino acid peptide within rpa43 that is both necessary and sufficient to mediate the interaction between rpa43 and Rrn3. This peptide inhibited rDNA transcription in vitro, while a control peptide did not. To determine the effect of the peptide in cultured cells, the peptide was coupled to the HIV TAT peptide to facilitate transduction into cells. The wild-type peptide, but not control peptides, inhibited Pol I transcription and cell division. In addition, the peptide induced cell death, consistent with other observations that "nucleolar stress" results in the death of tumor cells. The 22mer is a small-molecule inhibitor of rDNA transcription that is specific for the interaction between Rrn3 and rpa43, as such it represents an original way to interfere with cell growth. IMPLICATIONS: These results demonstrate a potentially novel pharmaceutical target for the therapeutic treatment of cancer cells.

Characterization of the interactions of mammalian RNA polymerase I associated proteins PAF53 and PAF49.

Masami Muramatsu's laboratory demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. They have also identified a second polymerase associated factor, PAF49. Both PAF49 and PAF53 copurify with that fraction of the RNA polymerase I molecules that can function in transcription initiation in vitro. PAF49 and PAF53 are the mammalian homologues of two subunits of yeast RNA polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex in yeast RNA polymerase I. In light of those publications, we investigated the interactions between various deletion and substitution mutants of mammalian PAF49 and PAF53 with the purpose of identifying those domains of the mammalian proteins that interact. Comparison of our results with structural studies on yeast A34.5 and A49 demonstrates that the yeast and mammalian proteins may in fact share structural similarities. In fact, the deletion mutagenesis data confirmed and extended the structural studies. For example, amino acids 41-86 of PAF49 were sufficient to provide the basis for heterodimerization. In silico structural analysis predicted that this region could assume a structure similar to the homologous region of yeast A34.5. Those similarities are insufficient, by themselves, for the proteins to form interspecific heterodimers. However, substitution of amino acids 52-98 of yeast A34.5 with amino acids 41-86 of mammalian PAF49 resulted in a protein that could heterodimerize with mouse PAF53.