Biology subjects

Lopez-Munoz, A. D.

Publications and source records attributed to Lopez-Munoz, A. D..

4 recordsLinked to original sources

bioRxiv2022

High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9

The application of CRISPR/Cas9 to improve genome engineering efficiency of large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desire locus has not been reported yet. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiency depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex viruses (HSV) type 1 and 2, to optimize a CRISPR/Cas9 method that can be versatilely used for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep-sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and color-based selection of recombinants. This application of CRISPR/Cas9 reduces time and labor of screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desire locus in less than two weeks. DATA SUMMARYRaw sequence reads are available at the European Bioinformatics Institute (EMBL-EBI) European Nucleotide Archive (ENA) as Bioproject ID PRJEB32151 and PRJEB32152. Six supplementary figures, eleven supplementary tables and supplementary methods are available with the online version of this article. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2021

Cell Surface SARS-CoV-2 Nucleocapsid Protein Modulates Innate and Adaptive Immunity

SARS-CoV-2 nucleocapsid protein (N) induces strong antibody and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2 infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12{beta}, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS CoV. Anti-N Abs bound to the surface of N expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.

microbiology↗

bioRxiv2021

Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants ability to potentially infect different animals. ImportanceSARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

microbiology↗

bioRxiv2021

Herpes simpex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue. AUTHOR SUMMARYHerpesviruses are highly human pathogens that establish latency in neurons of the peripheral nervous system. Colonization of nerve endings is required for herpes simplex virus (HSV) persistence and pathogenesis. HSV-1 global prevalence is much higher than HSV-2, in addition to their preferential tendency to infect the oronasal and genital areas, respectively. How these closely related viruses have been adapting and evolving to replicate and colonize these two different anatomical areas remains unclear. Herpesviruses were presumed to mutate much less than viruses with RNA genomes, due to the higher fidelity of the DNA polymerase and proofreading mechanisms when replicating. However, the worldwide accessibility and development of high-throughput sequencing technologies have revealed the heterogenicity and high diversity present in viral populations clinically isolated. Here we show that HSV-2 mutates much faster than HSV-1, when compared under similar and controlled cell culture conditions. This high mutation rate is translated into an increase in coding diversity, since the great majority of these new mutations lead to nonconservative changes in viral proteins. Understanding how herpesviruses differentially mutate under similar selective pressures is critical to prevent resistance to anti-viral drugs.

microbiology↗