Biology subjects

Kleinpeter, A. B.

Publications and source records attributed to Kleinpeter, A. B..

3 recordsLinked to original sources

bioRxiv2026

EZ-DMS - A Simple and Accessible Protocol and Software Package for Deep Mutational Scanning of Virus Proteins

Mutations that occur during viral genome replication can result in new phenotypes, such as infection of new cell types or resistance to therapeutics. To determine the phenotypic potential of viruses, deep mutational scanning (DMS) can be used. This approach measures the effects of all amino acid changes at sites of interest on protein function, virus infectivity, or resistance to therapeutics. A virus library that contains all possible variants is produced and used to infect a culture of cells. The frequency of each amino acid at the DMS site in the infected culture relative to the input stock captures the relative fitness for each form under the selective pressure applied. Current DMS protocols are complex and costly due to reliance on deep sequencing for genomic characterization. They also require expertise in bioinformatics to process and analyze the data, thus limiting the number of labs that can apply the technology. Here, we describe a simple and efficient protocol to perform DMS on single sites and on two-site combinations in the Env protein of HIV-1. Dual-site DMS allows probing for epistatic effects. The protocol greatly reduces recombination of the proviral genome in transformed bacteria, thus efficiency of the method. Sequencing is performed using Oxford Nanopore technology, and the data are uploaded to a graphical user interface-based platform that calculates the amino acid preferences, which describe the relative fitness of all amino acids under the experimental conditions tested (e.g., under selection pressure of a drug). The protocol does not require expertise in bioinformatics and can be completed in approximately 16 days, with 3-4 days of laboratory activity and 12-13 days of incubation, with considerably lower costs than existing systems.

microbiology↗

bioRxiv2025

The central pore of HIV-1 capsomers promotes sustained stability of the viral capsid

The HIV-1 capsid, which orchestrates several key post-entry events to facilitate infection in target cells, is composed of hexamers and pentamers (capsomers) of the capsid (CA) protein arranged in a closed, conical structure known as the capsid that protects the viral RNA genome and replicative enzymes reverse transcriptase (RT) and integrase (IN). Each capsomer contains a central pore lined with rings of positively charged amino acid side chains - Arg-18 (R18) and Lys-25 (K25). The R18 and K25 rings drive capsid assembly by binding the host polyanion inositol hexakisphosphate (IP6) and are proposed to mediate the import of dNTPs into the capsid to facilitate reverse transcription. Here we demonstrate that the R18 ring can be functionally replaced by the introduction of a mutation (N21K) that establishes a new electropositive ring within the central pore. In contrast with previous studies in which R18 mutants were unable to adapt in culture, the N21K mutation facilitated the acquisition of second-site compensatory mutations that restored near-WT fitness to viral mutants lacking the R18 ring. Comparative analysis of several central pore mutants lacking the R18 ring revealed that particle infectivity was not correlated with IP6 binding or capsid assembly, but rather with capsid stability and key post-entry events including reverse transcription and nuclear entry. Our results indicate that the central pore plays critical roles in both the assembly of capsids and their sustained stability post-entry.

microbiology↗

bioRxiv2020

CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

microbiology↗