Biology subjects

Poston, D.

Publications and source records attributed to Poston, D..

6 recordsLinked to original sources

bioRxiv2021

VPS29 exerts opposing effects on endocytic viral entry

Emerging zoonotic viral pathogens threaten global health and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic and pandemic threat coronaviruses as well as ebolavirus. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. VPS29 deficiency caused changes endosome morphology, and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence viral susceptibility and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases.

microbiology↗

bioRxiv2021

High genetic barrier to escape from human polyclonal SARS-CoV-2 neutralizing antibodies

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in SARS-CoV-2 convalescent and vaccinated individuals are key determinants of neutralization breadth and, consequently, the genetic barrier to viral escape. Using chimeric viruses and antibody-selected viral mutants, we show that multiple neutralizing epitopes, within and outside the viral receptor binding domain (RBD), are variably targeted by polyclonal plasma antibodies and coincide with sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic polymutant spike proteins that resisted polyclonal antibody neutralization to a similar degree as currently circulating variants of concern (VOC). Importantly, by aggregating VOC-associated and plasma-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in SARS-CoV-2 spike are sufficient to confer near-complete resistance to the polyclonal neutralizing antibodies generated by convalescents and mRNA vaccine recipients. Strikingly however, plasma from individuals who had been infected and subsequently received mRNA vaccination, neutralized this highly resistant SARS-CoV-2 polymutant, and also neutralized diverse sarbecoviruses. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against future sarbecovirus pandemics.

microbiology↗

bioRxiv2021

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

microbiology↗

bioRxiv2021

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

microbiology↗

bioRxiv2021

Derivation and Characterization of an inositol phosphate-independent HIV-1

A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation is facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6-deficiency, we passaged HIV-1 mutants that had substitutions in IP6-coordinating residues to select for compensatory mutations. We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image the BVM-inducible assembly of individual HIV-1 particles assembly in living cells. Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can functionally replace IP6. Author SummaryA key step in HIV-1 replication is the assembly of virions that are released from the infected cell. Previous work has suggested that a small molecule called IP6 is critical role in this process, promoting both HIV-1 assembly and the stability of mature fully infectious virions. Since IP6 is required for multiple steps in HIV-1 assembly and maturation, it is a candidate for the development of anti-retroviral therapies. Here, we identify an HIV-1 mutant that replicates independently of IP6, and show that a different small molecule can functionally substitute for IP6 under certain conditions. These findings suggest that IP6 regulates the stability of protein interactions during virion assembly and that the precise degree stability of these interactions is finely tuned and important for generating infectious virions. Finally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

microbiology↗

bioRxiv2020

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

microbiology↗