Biology subjects

Burnett, C.

Publications and source records attributed to Burnett, C..

6 recordsLinked to original sources

bioRxiv2026

Structural Basis for Dimeric Genome Selection by HIV-1

HIV-1 packages a dimeric RNA genome into assembling virions, a requirement for replication. To understand the mechanism of dimer selection, we conducted cryogenic electron microscopy (cryoEM) studies of nascent assemblies formed between HIV-1 Gag protein constructs and genomic RNA elements that promote dimerization and packaging. CryoEM maps revealed structural architectures in which the helical dimer initiation signal (DIS) of the RNA packaging signal bridges two 6-helix bundles (6HBs) that protrude from adjacent Gag hexamers. Conserved guanosines that sandwich the DIS are located beneath the 6HBs and poised to interact with clusters of nucleocapsid domains. Mutation of these guanosines severely impairs RNA packaging into virus-like particles. Our findings suggest that DIS functions as a molecular ruler, organizing Gag hexamers into structures optimized for Gag lattice expansion.

molecular biology↗

bioRxiv2024

HIV-1 single transcription start site mutants display complementary replication functions that are restored by reversion

HIV-1 transcription initiates at two positions, generating RNAs with either cap1G or cap3G 5' ends. The replication fates of these RNAs differ, with viral particles encapsidating almost exclusively cap1G RNAs and cap3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection. Results showed that either single start RNA could serve as both mRNA and genomic RNA when present as the only form in cells, although cap3G RNA was more efficiently translated and spliced while cap1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, cap1G RNA was preferentially packaged into virions. During spreading infection cap1G-only virus displayed only minor defects but cap3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of cap3G-only virus yielded revertants that replicated as well as the twinned (cap1G+ cap3G) transcription start site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start site use. ImportanceHIV-1 generates two RNAs during its replication that differ by only two nucleotides in length. Despite this very minor difference, the RNAs perform different and complementary replication functions. When mutants that expressed only one RNA were forced to revert, they regained functions associated with the second RNA.

microbiology↗

bioRxiv2023

Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5 isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogenous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs. Phylogenetic csomparisons and chimeric viruses properties provided evidence that this mechanism of RNA fate determination was an innovation of the HIV-1 lineage, with determinants mapping to core promoter elements. Fine-tuning differences between HIV-1 and HIV-2, which uses a unique TSS, implicated purine residue positioning plus a specific TSS-adjacent dinucleotide in specifying multiplicity of TSS usage. Based on these findings, HIV-1 expression constructs were generated that differed from the parental strain by only two point mutations yet each expressed only one of HIV-1s two RNAs. Replication defects of the variant with only the presumptive founder TSS were less severe than those for the virus with only the secondary start site.

molecular biology↗

bioRxiv2021

Vpr shapes the proviral landscape and polyclonal HIV-1 reactivation patterns in cultured cells

Cell culture models suggest that the HIV-1 viral protein R (Vpr) is dispensable for latency establishment. However, whether Vpr affects the persistent proviral landscape and responsiveness to latency reversing agents (LRAs) is unclear. Here, integration site landscape, clonal dynamics, and latency reversal effects of Vpr were studied by comparing barcoded vpr+ and vpr- populations arising after infection of Jurkat cells in vitro. The results showed that individual integrant clones differed in fractions of LTR-active daughter cells: some clones gave rise to few to no LTR-active cells while for others almost all daughter cells were LTR-active. Integrant clones with at least 60% LTR-active cells (high LTR-active clones) contained proviruses positioned closer to preexisting enhancers (H3K27ac) and promoters (H3K4me3) than clones with <30% LTR-active cells (low LTR-active clones). Comparing vpr+ and vpr- populations revealed that the vpr+ population was depleted of high LTR-active clones. Complementing vpr-defective proviruses by transduction with vpr 16 days after infection led to rapid loss of high LTR-active clones, indicating that the effect of Vpr on proviral populations occurs post-integration. Comparing vpr+ and vpr- integration sites revealed that predominant vpr+ proviruses were farther from enhancers and promoters. Correspondingly, distances to these marks among previously reported intact HIV proviruses in ART-suppressed patients were more similar to those in the vpr+ pool than to vpr- integrants. To compare latency reactivation agent (LRA) responsiveness, the LRAs prostratin and JQ1 were applied separately or in combination. vpr+ and vpr- population-wide trends were similar, but combination treatment reduced virion release in a subset of vpr- clones relative to when JQ1 was applied separately, an effect not observed in vpr+ pools. Together, these observations highlight the importance of Vpr to proviral population dynamics, integration site landscapes, and responsiveness to latency reversing agents. One Sentence Summary Expression properties and responsiveness to latency reactivation agents of individual HIV-1 proviral clones within polyclonal populations are masked by dominant clones and influenced by proviral proximity to certain epigenetic marks and by Vpr, a viral factor not previously known to affect latency and reactivation.

microbiology↗

bioRxiv2020

High-resolution yeast quiescence profiling in human-like media reveals interacting influences of auxotrophy and nutrient availability

Yeast cells survive in stationary phase culture by entering quiescence, which is measured by colony forming capacity upon nutrient re-exposure. Yeast chronological lifespan (CLS) studies, employing the comprehensive collection of gene knockout strains, have correlated weakly between independent laboratories, which is hypothesized to reflect differential interaction between the deleted genes, auxotrophy, media composition and other assay conditions influencing quiescence. This hypothesis was investigated by high-throughput quiescence profiling of the parental prototrophic strain, from which the gene deletion strain libraries were constructed, and all possible auxotrophic allele combinations in that background. Defined media resembling human cell culture media promoted long-term quiescence, and was used to assess effects of glucose, ammonium sulfate, auxotrophic nutrient availability, Target of Rapamycin signaling, and replication stress. Frequent, high-replicate measurements of colony forming capacity from cultures aged past 60 days provided profiles of quiescence phenomena such as gasping and hormesis. Media acidification was assayed in parallel to assess correlation. Influences of leucine, methionine, glucose, and ammonium sulfate metabolism were clarified, and a role for lysine metabolism newly characterized, while histidine and uracil perturbations had less impact. Interactions occurred between glucose, ammonium sulfate, auxotrophy, auxotrophic nutrient limitation, aeration, TOR signaling, and/or replication stress. Weak correlation existed between media acidification and maintenance of quiescence. In summary, experimental factors, uncontrolled across previous genome-wide yeast CLS studies, influence quiescence and interact extensively, revealing quiescence as a complex metabolic and developmental process that should be studied in a prototrophic context, omitting ammonium sulfate from defined media, and employing highly replicable protocols.

genetics↗

bioRxiv2019

Enabling MHC-mismatched hematopoietic stem cell transplants and organ graft tolerance without chemotherapy or radiation

Hematopoietic stem cell (HSC) transplantation can replace diseased blood systems with a healthy one, thereby treating or curing genetic blood and immune disorders including autoimmune diseases and immunodeficiencies. However, toxic chemotherapy or radiation is necessary to ablate an animals existing blood system prior to HSC transplantation, leading to significant morbidity. To accomplish safer blood-system replacement we developed a combination of six monoclonal antibodies to safely and specifically deplete the HSCs, T cells and NK cells of immune-competent mice. Remarkably, immunologically-foreign (allogeneic) HSCs mismatched at half or all the MHC genes could engraft these antibody-treated mice, generating donor blood systems that stably co-existed with host blood cells. These chimeric immune systems were immunologically tolerant to heart tissue from the HSC donor, providing a safe platform for HSC transplantation as a means to solid organ transplantation. The ability to transplant MHC-mismatched HSCs without chemotherapy or radiation has significant ramifications for regenerative medicine.

immunology↗