Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

HexSE: Simulating evolution in overlapping reading frames.

Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may provide a mechanism to increase the information content of compact genomes. The presence of overlapping reading frames (OvRFs) can skew estimates of selection based on the rates of non-synonymous and synonymous substitutions, since a substitution that is synonymous in one reading frame may be non-synonymous in another and vice versa. To understand the impact of OvRFs on molecular evolution, we implemented a versatile simulation model of nucleotide sequence evolution along a phylogeny with any distribution of open reading frames in linear or circular genomes. We use a custom data structure to track the substitution rates at every nucleotide site, which is determined by the stationary nucleotide frequencies, transition bias and the distribution of selection biases (dN/dS) in the respective reading frames. Our simulation model is implemented in the Python scripting language. All source code is released under the GNU General Public License version 3 and are available at https://github.com/PoonLab/HexSE.

Clustering Highly Divergent Homologous Proteins: An Alignment-Free Method.

The comparative analysis of amino acid sequences is an important tool in molecular biology that often requires multiple sequence alignments. In comparisons between less closely related genomes, however, it becomes more difficult to accurately align protein-coding sequences, or even to identify homologous regions in different genomes. In this article, we describe an alignment-free method for the classification of homologous protein-coding regions from different genomes. This methodology was originally developed for comparing genomes within virus families, but may be adapted for other organisms. We quantify sequence homology from the overlap (intersection distance) of the k-mer (word) frequency distributions for different protein sequences. Next, we extract groups of homologous sequences from the resulting distance matrix using a combination of dimensionality reduction and hierarchical clustering methods. Finally, we demonstrate how to generate visualizations of the composition of clusters with respect to protein annotations, and by coloring protein-coding regions of genomes by cluster assignments. These provide a useful means to quickly assess the reliability of the clustering results based on the distribution of homologous genes among genomes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data collection and processing Basic Protocol 2: Calculating k-mer distances Basic Protocol 3: Extracting clusters of homology Support Protocol: Genome plot based on clustering results.

Using networks to analyze and visualize the distribution of overlapping genes in virus genomes.

Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may increase the information content of compact genomes or influence the creation of new genes. Here we report a global comparative study of overlapping open reading frames (OvRFs) of 12,609 virus reference genomes in the NCBI database. We retrieved metadata associated with all annotated open reading frames (ORFs) in each genome record to calculate the number, length, and frameshift of OvRFs. Our results show that while the number of OvRFs increases with genome length, they tend to be shorter in longer genomes. The majority of overlaps involve +2 frameshifts, predominantly found in dsDNA viruses. Antisense overlaps in which one of the ORFs was encoded in the same frame on the opposite strand (-0) tend to be longer. Next, we develop a new graph-based representation of the distribution of overlaps among the ORFs of genomes in a given virus family. In the absence of an unambiguous partition of ORFs by homology at this taxonomic level, we used an alignment-free k-mer based approach to cluster protein coding sequences by similarity. We connect these clusters with two types of directed edges to indicate (1) that constituent ORFs are adjacent in one or more genomes, and (2) that these ORFs overlap. These adjacency graphs not only provide a natural visualization scheme, but also a novel statistical framework for analyzing the effects of gene- and genome-level attributes on the frequencies of overlaps.

Inheritance of Rootstock Effects in Avocado (Persea americana Mill.) cv. Hass.

Grafting is typically utilized to merge adapted seedling rootstocks with highly productive clonal scions. This process implies the interaction of multiple genomes to produce a unique tree phenotype. However, the interconnection of both genotypes obscures individual contributions to phenotypic variation (rootstock-mediated heritability), hampering tree breeding. Therefore, our goal was to quantify the inheritance of seedling rootstock effects on scion traits using avocado (Persea americana Mill.) cv. Hass as a model fruit tree. We characterized 240 diverse rootstocks from 8 avocado cv. Hass orchards with similar management in three regions of the province of Antioquia, northwest Andes of Colombia, using 13 microsatellite markers simple sequence repeats (SSRs). Parallel to this, we recorded 20 phenotypic traits (including morphological, biomass/reproductive, and fruit yield and quality traits) in the scions for 3 years (2015-2017). Relatedness among rootstocks was inferred through the genetic markers and inputted in a "genetic prediction" model to calculate narrow-sense heritabilities (h 2) on scion traits. We used three different randomization tests to highlight traits with consistently significant heritability estimates. This strategy allowed us to capture five traits with significant heritability values that ranged from 0.33 to 0.45 and model fits (r) that oscillated between 0.58 and 0.73 across orchards. The results showed significance in the rootstock effects for four complex harvest and quality traits (i.e., total number of fruits, number of fruits with exportation quality, and number of fruits discarded because of low weight or thrips damage), whereas the only morphological trait that had a significant heritability value was overall trunk height (an emergent property of the rootstock-scion interaction). These findings suggest the inheritance of rootstock effects, beyond root phenotype, on a surprisingly wide spectrum of scion traits in "Hass" avocado. They also reinforce the utility of polymorphic SSRs for relatedness reconstruction and genetic prediction of complex traits. This research is, up to date, the most cohesive evidence of narrow-sense inheritance of rootstock effects in a tropical fruit tree crop. Ultimately, our work highlights the importance of considering the rootstock-scion interaction to broaden the genetic basis of fruit tree breeding programs while enhancing our understanding of the consequences of grafting.

SECUENCIACIÓN DEL GENOMA COMPLETO DEL Potato yellow vein virus (PYVV) EN TOMATE (Solanum lycopersicum) EN COLOMBIA / Genome Sequencing of Potato yellow vein virus (PYVV) Strain Infecting Solanum lycopersicum in Colombia

RESUMEN Potato yellow vein virus (PYVV), es uno de los fitopatógenos más limitantes para la producción de papa en la región de Los Andes. A pesar que se le ha detectado infectando tomate en Colombia, el conocimiento de las características biológicas de las cepas presentes en este hospedante es muy limitado. En este estudio, utilizando secuenciación masiva de nueva generación (NGS), se obtuvo la secuencia completa de los tres segmentos genómicos del PYVV en plantas de tomate en Marinilla (Antioquia) y se evalúo la utilidad de tres juegos de cebadores para su detección mediante pruebas de RT-PCR convencional y en tiempo real (RT-qPCR). El genoma de la secuencia consenso presentó tamaños de 8043 nt (ARN1), 5346 nt (ARN2) y 3896 nt (ARN3) y se identificaron los diez ORF previamente reportados en este virus, aunque, en general, éstos presentaron menores niveles de identidad que los registrados entre cepas de PYVV de papa. Análisis de variación y de selección identificaron dos regiones en los ORF MET/HEL y CPm que presentan selección positiva, lo que podría estar asociado a la adaptación por hospedante. Los tres juegos de cebadores amplificaron las regiones esperadas de la cápside de PYVV, siendo posible identificar, por diferencias en valores de temperatura de fusión (Tm) y por secuenciación Sanger, la ocurrencia de al menos dos variantes principales de este virus en el Oriente Antioqueño, lo que concuerda con los niveles moderados de polimorfismos encontrados en las secuencias obtenidas por NGS.

ABSTRACT Potato yellow vein virus (PYVV) is one of the most important pathogens of potato in the Andean region. In spite of having been detected in tomato crops in Colombia, knowledge on the biological characteristics of PYVV is limited on this host. In this study, next-generation sequencing (NGS) of a PYVV strain infecting tomato in Marinilla District (Antioquia) was performed; additionally, three primer set useful in RT-PCR and RT-qPCR detection were also tested. The consensus genome consisted of three RNA segments of 8043 nt (RNA1), 5346 nt (RNA2) and 3896 nt (RNA3) encoding ten ORF with slight lower sequence identity in relation to PYVV isolates from potato. Sequence analysis suggests the presence of regions potentially undergoing positive selection in the ORFs coding for MET/HEL and CPm possibly as a result of host adaptation. Experimental validation of primers resulted in amplicon with the expected size while melting temperature analysis and sequencing suggest the presence of at least two PYVV infecting S. lycopersicum in east Antioquia in agreement with the NGS data.