The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription

Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization.

Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1–neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined “neutralization fingerprints” for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1–infected donors and a chimeric siman-human immunodeficiency virus–infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies

Novel Molecular Insights Into Epstein-Barr Virus Reactivation

Epstein-Barr Virus (EBV) is a herpesvirus responsible for approximately 1% of cancers worldwide, including African Burkitt lymphoma, Hodgkin lymphoma, lymphomas in immunosuppressed patients, and nasopharyngeal and gastric carcinoma. Interestingly, the development of different cancers is mainly due to expression of the latent EBV proteins, although expression of lytic genes has recently been shown to play a role. Many of the molecular mechanisms behind EBV reactivation have been revealed, however, the contribution of chromatin dynamics to EBV reactivation is not well understood. A better understanding of how the switch between the latent and lytic cycle is regulated could have implications in treating disease. Here we investigate the contribution of chromatin dynamics to EBV reactivation in the context of i) nuclear localization and contacts with the human genome, and ii) the chromatin-reading protein BRD4. We used in situ Hi-C to show that the Epstein-Barr virus associates with repressive compartments of the nucleus during latency, and non-repressive compartments of the nucleus during reactivation. This adds 3D re-localization as a novel component to the molecular events that occur during EBV reactivation. Furthermore, we show that the protein BRD4 plays an important role in EBV lytic reactivation. BRD4 binds to the lytic origins of replication and inhibition of BRD4 by JQ1 inhibits the lytic cycle at two different steps. In summary, this work has led to a better understanding of how the latent-lytic switch of EBV is regulated

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors

The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription

Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease.

Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) in a yeast screen. We identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir, many of which have not been previously reported. Ninety-nine mutations caused apparent resistance to both inhibitors, suggesting likelihood for the evolution of cross-resistance. The mutation with the strongest drug resistance score against nirmatrelvir in our study (E166V) was the most impactful resistance mutation recently reported in multiple viral passaging studies. Many mutations that exhibited inhibitor-specific resistance were consistent with the distinct interactions of each inhibitor in the substrate binding site. In addition, mutants with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct-resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and compensatory mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations

Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance.

Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms.

With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance

Localized protein biotinylation at DNA damage sites identifies ZPET, a repressor of homologous recombination

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins