Fabio Zanini

Population genomics of intrapatient HIV-1 evolution

Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. DOI: http://dx.doi.org/10.7554/eLife.11282.001

Viscosity and diffusion: crowding and salt effects in protein solutions

We report on a joint experimental–theoretical study of collective diffusion in, and static shear viscosity of solutions of bovine serum albumin (BSA) proteins, focusing on the dependence on protein and salt concentration. Data obtained from dynamic light scattering and rheometric measurements are compared to theoretical calculations based on an analytically treatable spheroid model of BSA with isotropic screened Coulomb plus hard-sphere interactions. The only input to the dynamics calculations is the static structure factor obtained from a consistent theoretical fit to a concentration series of small-angle X-ray scattering (SAXS) data. This fit is based on an integral equation scheme that combines high accuracy with low computational cost. All experimentally probed dynamic and static properties are reproduced theoretically with an at least semi-quantitative accuracy. For lower protein concentration and low salinity, both theory and experiment show a maximum in the reduced viscosity, caused by the electrostatic repulsion of proteins. On employing our theoretical and experimental results, the applicability range of a generalized Stokes–Einstein (GSE) relation connecting viscosity, collective diffusion coefficient, and osmotic compressibility, proposed by Kholodenko and Douglas [Phys. Rev. E, 1995, 51, 1081] is examined. Significant violation of the GSE relation is found, both in experimental data and in theoretical models, in concentrated systems at physiological salinity, and under low-salt conditions for arbitrary protein concentrations.

Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution

ABSTRACT Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8+ T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not.

FFPopSim: an efficient forward simulation package for the evolution of large populations

Motivation: The analysis of the evolutionary dynamics of a population with many polymorphic loci is challenging, as a large number of possible genotypes needs to be tracked. In the absence of analytical solutions, forward computer simulations are an important tool in multi-locus population genetics. The run time of standard algorithms to simulate sexual populations increases as 8L with the number of loci L, or with the square of the population size N. Results: We have developed algorithms to simulate large populations with arbitrary genetic maps, including multiple crossovers, with a run time that scales as 3L. If the number of crossovers is restricted to at most one, the run time is reduced to L2L. The algorithm is based on an analogue of the Fast Fourier Transform (FFT) and allows for arbitrary fitness functions (i.e. any epistasis). In addition, we include a streamlined individual-based framework. The library is implemented as a collection of C++ classes and a Python interface. Availability: http://code.google.com/p/ffpopsim/. Contact: richard.neher@tuebingen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

RuvB-like ATPases function in chromatin decondensation at the end of mitosis.

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

Thalidomide Mimics Uridine Binding to an Aromatic Cage in Cereblon.

Thalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.

Establishment and stability of the latent HIV-1 DNA reservoir

HIV-1 infection cannot be cured because the virus persists as integrated proviral DNA in long-lived cells despite years of suppressive antiretroviral therapy (ART). In a previous paper (Zanini et al, 2015) we documented HIV-1 evolution in 10 untreated patients. Here we characterize establishment, turnover, and evolution of viral DNA reservoirs in the same patients after 3–18 years of suppressive ART. A median of 14% (range 0–42%) of the DNA sequences were defective due to G-to-A hypermutation. Remaining DNA sequences showed no evidence of evolution over years of suppressive ART. Most sequences from the DNA reservoirs were very similar to viruses actively replicating in plasma (RNA sequences) shortly before start of ART. The results do not support persistent HIV-1 replication as a mechanism to maintain the HIV-1 reservoir during suppressive therapy. Rather, the data indicate that DNA variants are turning over as long as patients are untreated and that suppressive ART halts this turnover. DOI: http://dx.doi.org/10.7554/eLife.18889.001

Error rates, PCR recombination, and sampling depth in HIV-1 whole genome deep sequencing

Deep sequencing is a powerful and cost-effective tool to characterize the genetic diversity and evolution of virus populations. While modern sequencing instruments readily cover viral genomes many thousand fold and very rare variants can in principle be detected, sequencing errors, amplification biases, and other artifacts can limit sensitivity and complicate data interpretation. For this reason, the number of studies using whole genome deep sequencing to characterize viral quasi-species in clinical samples is still limited. We have previously undertaken a large scale whole genome deep sequencing study of HIV-1 populations. Here we discuss the challenges, error profiles, control experiments, and computational test we developed to quantify the accuracy of variant frequency estimation.

Salt-Induced Universal Slowing Down of the Short-Time Self-Diffusion of a Globular Protein in Aqueous Solution

The short-time self-diffusion D of the globular model protein bovine serum albumin in aqueous (D2O) solutions has been measured comprehensively as a function of the protein and trivalent salt (YCl3) concentration, noted cp and cs, respectively. We observe that D follows a universal master curve D(cs,cp) = D(cs = 0,cp) g(cs/cp), where D(cs = 0,cp) is the diffusion coefficient in the absence of salt and g(cs/cp) is a scalar function solely depending on the ratio of the salt and protein concentration. This observation is consistent with a universal scaling of the bonding probability in a picture of cluster formation of patchy particles. The finding corroborates the predictive power of the description of proteins as colloids with distinct attractive ion-activated surface patches.

In-vivo mutation rates and fitness landscape of HIV-1

Mutation rates and fitness costs of deleterious mutations are difficult to measure in vivo but essential for a quantitative understanding of evolution. Using whole genome deep sequencing data from longitudinal samples during untreated HIV-1 infection, we estimated mutation rates and fitness costs in HIV-1 from the temporal dynamics of genetic variation. At approximately neutral sites, mutations accumulate with a rate of 1.2 × 10−5 per site per day, in agreement with the rate measured in cell cultures. The rate from G to A is largest, followed by the other transitions C to T, T to C, and A to G, while transversions are more rare. At non-neutral sites, most mutations reduce virus replication; using a model of mutation selection balance, we estimated the fitness cost of mutations at every site in the HIV-1 genome. About half of all nonsynonymous mutations have large fitness costs (greater than 10%), while most synonymous mutations have costs below 1%. The cost of synonymous mutations is especially low in most of gag and pol, while much higher costs are observed in important RNA structures and regulatory regions. The intrapatient fitness cost estimates are consistent across multiple patients, suggesting that the deleterious part of the fitness landscape is universal and explains a large fraction of global HIV-1 group M diversity.