Biology subjects

Vashee, S.

Publications and source records attributed to Vashee, S..

6 recordsLinked to original sources

bioRxiv2026

Modular Assembly of an Infectious Clone of the Akata strain of EBV using Synthetic Genomics Methods in Yeast

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Synthetic Biology↗

bioRxiv2024

The loss of both pUL16 and pUL21 in HSV-1 infected cells abolishes cytoplasmic envelopment.

Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1). To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation associated recombination. The eleven fragments or "parts" spanned the 152 kb genome and recombination was achieved because of the overlapping homologous sequences between each fragment. To demonstrate the robustness of this genome assembly method for reverse genetics, we engineered different mutations that were located in distant loci on the genome and built a collection of HSV-1 genomes that contained single and different combination of mutations in 5 conserved HSV-1 genes. The five genes: UL7, UL11, UL16, UL21 and UL51 encode virion structural proteins and have varied functions in the infected cell. Each is dispensable for virus replication in cell culture, however, combinatorial analysis of deletions in the five genes revealed "synthetic-lethality" of some of the genetic mutations. Thus, it was discovered that any virus that carried a UL21 mutation in addition to the other gene was unable to replicate in Vero cells. Replication was restored in a complementing cell line that provided pUL21 in trans. One particular combination (UL16-UL21) was of interest because the proteins encoded by these genes are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1 infected cells are unclear. Both are dispensable for HSV-1 replication, however, in HSV-2 their mutation results in nuclear retention of assembled capsids. We thus characterized these viruses that carry the single and double mutant. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident but did not mature into enveloped particles. The capsid particles isolated from these cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the wild-type capsids. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16; the complex of pUL16 and pUL21 should be considered as important mediators of cytoplasmic maturation of the particle.

microbiology↗

bioRxiv2024

Multiomic Insights into Human Health: Gut Microbiomes of Hunter-Gatherer, Agropastoral, and Western Urban Populations

Societies with exposure to preindustrial diets exhibit improved markers of health. Our study used a comprehensive multi-omic approach to reveal that the gut microbiome of the Ju/hoansi hunter-gatherers, one of the most remote KhoeSan groups, exhibit a higher diversity and richness, with an abundance of microbial species lost in the western population. The Ju/hoansi microbiome showed enhanced global transcription and enrichment of complex carbohydrate metabolic and energy generation pathways. The Ju/hoansi also show high abundance of short-chain fatty acids that are associated with health and optimal immune function. In contrast, these pathways and their respective species were found in low abundance or completely absent in Western populations. Amino acid and fatty acid metabolism pathways were observed prevalent in the Western population, associated with biomarkers of chronic inflammation. Our study provides the first in-depth multi-omic characterization of the Ju/hoansi microbiome, revealing uncharacterized species and functional pathways that are associated with health.

genomics↗

bioRxiv2023

SARS-CoV-2 ORF8 modulates lung inflammation and clinical disease progression

The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis. SignificanceSince its emergence in 2019, SARS-CoV-2 has accrued mutations throughout its 30kb genome. Of particular interest are the mutations present in the ORF8 protein, which occur in every major variant. The precise function and impact of this protein on disease severity and pathogenesis remains understudies. Our studies reveal that the ORF8 protein modulates the immune response by impacting macrophage infiltration into the lungs. Additionally, we have shown that the ORF8 protein of SARS-CoV-2 has accrued mutations throughout its evolution that lead to a loss of function phenotype in this protein. Our work reveals that the ORF8 protein of SARS-CoV-2 contributes significantly to disease progression through modulation of the inflammatory response.

microbiology↗

bioRxiv2022

SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. SignificanceA hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.

microbiology↗

bioRxiv2019

Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri (Mmc) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately ten percent of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually nonessential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though ten percent of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reaching endpoint criteria within seven days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.\n\nIMPORTANCEMembers of the Mycoplasma mycoides cluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools to Mycoplasma mycoides in order to design and create a fully attenuated Mycoplasma strain that was subsequently confirmed in vivo using a novel caprine infection model. This is the first time that a Mycoplasma mutant developed by applying synthetic biology tools has been tested in vivo to pin point candidate virulence traits. The mutant strain is similar to \"apathogenic E. coli K12\" strains that boosted the research on host-pathogen interactions for the genus Escherichia and other bacterial genera.

microbiology↗