Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind a2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also identify recent viral strains with reduced neutralization to sera elicited by candidate vaccine virus. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive characterization of mutations to inform surveillance of H5 influenza.

Mutations induced by Bleomycin, 4-nitroquinoline-1-oxide, and hydrogen peroxide in the rpoB gene of Escherichia coli: Perspective on Mutational Hotspots.

We report the mutational spectra in a segment of the E. coli rpoB gene of bleomycin (BLEO), 4-nitroquinoline-1-oxide (NQO), and hydrogen peroxide (H2O2). We compare these spectra with those of other mutagens and repair deficient strains in the same rpoB system, and review the key elements determining mutational hotspots and outline the questions that remain unanswered. We consider three tiers of hotspots that derive from 1) the nature of the sequence change at a specific base, 2) the direct nearest neighbors and 3) some aspect of the larger sequence context or the local 3D-structure of segments of DNA. This latter tier can have a profound effect on mutation frequencies, even among sites with identical nearest neighbor sequences. BLEO is dependent on the SOS-induced translesion Pol V for mutagenesis, and has a dramatic hotspot at a single mutational site in rpoB. NQO is not dependent on any of the translesion polymerases, in contrast to findings with plasmids treated in vitro and transformed into E. coli. The rpoB system allows one to monitor both G:C -> A:T transitions and G:C -> T:A transversions at the same site in 11 cases, each site having the identical sequence context for each of the two mutations. The combined preference for G:C -> A:T transitions at these sites is 20-fold. Several of the favored sites for hydrogen peroxide mutagenesis are not seen in the spectra of BLEO and NQO mutations, indicating that mutagenesis from reactive oxygen species is not a major cause of BLEO or NQO mutagenesis, but rather specific adducts. The variance in mutation rates at sites with identical nearest neighbors suggests that the local structure of different DNA segments is an important factor in mutational hotspots.

Capsid-CPSF6 interaction: Master regulator of nuclear HIV-1 positioning and integration.

HIV-1 integration favors active chromatin, which is primarily mediated through interactions between the viral capsid and integrase proteins with host factors cleavage and polyadenylation specificity factor 6 (CPSF6) and lens epithelium-derived growth factor/p75, respectively. Previously published image-based studies had suggested that HIV-1 prefers to integrate into chromatin that associates spatially with the nuclear periphery. Here, we re-evaluated previously reported HIV-1 nuclear distance measures across studies and show that HIV-1 prefers peri-nuclear and mid-nuclear zones similarly, with a common preference between studies mapping to the boundary between these two radial areas. We also discuss emerging roles for the capsid-CPSF6 interaction in facilitating HIV-1 pre-integration complex nuclear import and subsequent intranuclear trafficking to preferred sites of viral DNA integration.