Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Reconstruction of the cell entry pathway of an extinct virus.

Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERV-K Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.

A broad antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that fails to protect against both seasonal antigenic variants and the next pandemic. However, antibodies directed to conserved sites can confer broad protection. We identify and characterize a class of human antibodies that engage a previously undescribed, conserved, epitope on the influenza hemagglutinin protein (HA). Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal infection in mice. Their breadth of binding extends across most influenza A serotypes and seasonal human variants. Antibodies to the head-stem interface epitope are present at low frequency in the memory B cell populations of multiple donors. The immunogenicity of the epitope warrants its consideration for inclusion in improved or "universal" influenza vaccines.

Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape.

Zoonotic pandemics, such as that caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can follow the spillover of animal viruses into highly susceptible human populations. The descendants of these viruses have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses. In the spike glycoprotein, we found that recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.

A Prevalent Focused Human Antibody Response to the Influenza Virus Hemagglutinin Head Interface.

Novel animal influenza viruses emerge, initiate pandemics, and become endemic seasonal variants that have evolved to escape from prevalent herd immunity. These processes often outpace vaccine-elicited protection. Focusing immune responses on conserved epitopes may impart durable immunity. We describe a focused, protective antibody response, abundant in memory and serum repertoires, to a conserved region at the influenza virus hemagglutinin (HA) head interface. Structures of 11 examples, 8 reported here, from seven human donors demonstrate the convergence of responses on a single epitope. The 11 are genetically diverse, with one class having a common, IGκV1-39, light chain. All of the antibodies bind HAs from multiple serotypes. The lack of apparent genetic restriction and potential for elicitation by more than one serotype may explain their abundance. We define the head interface as a major target of broadly protective antibodies with the potential to influence the outcomes of influenza virus infection. IMPORTANCE The rapid appearance of mutations in circulating human influenza viruses and selection for escape from herd immunity require prediction of likely variants for an annual updating of influenza vaccines. The identification of human antibodies that recognize conserved surfaces on the influenza virus hemagglutinin (HA) has prompted efforts to design immunogens that might selectively elicit such antibodies. The recent discovery of a widely prevalent antibody response to the conserved interface between two HA "heads" (the globular, receptor-binding domains at the apex of the spike-like trimer) has added a new target for these efforts. We report structures of eight such antibodies, bound with HA heads, and compare them with each other and with three others previously described. Although genetically diverse, they all converge on a common binding site. The analysis here can guide immunogen design for preclinical trials.

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Structure of the Receptor Binding Domain of EnvP(b)1, an Endogenous Retroviral Envelope Protein Expressed in Human Tissues.

EnvP(b)1 is an endogenous retroviral envelope gene found in human and other primate genomes. We report EnvP(b)1 sequences in primate genomes consistent with an integration event between 40 and 71 million years ago. Using a highly specific polyclonal antiserum raised against the putative receptor binding domain (RBD) of human EnvP(b)1, we detected expression in human placenta, ovaries, and thymus. We found that EnvP(b)1 is proteolytically processed, and using cell-cell fusion assays in multiple primate cell lines, we demonstrated that extant EnvP(b)1 proteins from a variety of primate genomes are fusogenic. This work supports the idea that EnvP(b)1 is under purifying selection and its fusogenic activity has been maintained for over 40 million years. We determined the structure of the RBD of human EnvP(b)1, which defines structural similarities with extant leukemia viruses, despite little sequence conservation. This structure highlights a common scaffold from which novel receptor binding specificities likely evolved. The evolutionary plasticity of this domain may underlie the diversity of related Envs in circulating viruses.IMPORTANCE Organisms can access genetic and functional novelty by capturing viral elements within their genomes, where they can evolve to drive new cellular or organismal processes. We demonstrate that a retroviral envelope gene, EnvP(b)1, has been maintained and its fusion activity preserved for 40 to 71 million years. It is expressed as a protein in multiple healthy human tissues. We determined the structure of its inferred receptor binding domain and compared it with the same domain in modern viruses. We found a common conserved architecture that underlies the varied receptor binding activity of divergent Env genes. The modularity and versatility of this domain may underpin the evolutionary success of this clade of fusogens.