A tool to probe SARS-CoV-2 biology To develop therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emerging variants, it is important to understand the viral biology and the effect of mutations. However, this is challenging because live virus can only be studied in a few laboratories that meet stringent safety standards. Syed et al . describe a virus-like particle (VLP) that comprises the four SARS-CoV-2 structural proteins, but instead of packaging viral RNA, it packages messenger RNA (mRNA) that expresses a reporter protein (see the Perspective by Johnson and Menachery). The amount of reporter expressed in receiver cells depends on the efficiency of packaging and assembly in the producer cells and the efficiency of entry into receiver cells. Mutations in the nucleocapsid protein that are found in more transmissible variants increase mRNA packaging and expression. The VLPs provide a platform for studying the effect of mutations in the structural proteins and for screening therapeutics. —VV

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M).

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform–containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.

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In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA upon PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

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Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose expression is associated with TERT reactivation and telomere maintenance. Here, using biochemical and cell biology approaches, we show direct evidence that a specific GABP complex containing the subunit protein GABPB1L forms predominantly at the mutant TERT promoter, leading to TERT re-expression. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, are immediately dependent on GABPB1L for proliferation in cell culture and post-tumor establishment in vivo. Notably, when combined with frontline temozolomide (TMZ) chemotherapy, GABPB1L knockdown and the associated TERT reduction lead to an impaired DNA damage response that results in profoundly reduced growth of intracranial GBM tumors.Together, these findings provide new insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of TERT suppression in GBM maintenance, and establish GABPB1L inhibition, alone or in combination with chemotherapy, as a therapeutic strategy for TERTp mutant GBM.