Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract\ud \ud \ud \ud Background\ud The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression.\ud \ud \ud \ud Results\ud We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization.\ud \ud \ud \ud Conclusions\ud Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.JVCO, CTB and AI hold FAPESP scholarships (04/12456-0, 09/16740-8 and 12/04818-5), AFB and CCMF hold CAPES-MSc and PhD scholarships and PMAZ holds a CNPq-PQ scholarship. This work was supported financially by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, process: 2007/55282-0)

A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.Fil: López, M. Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vinzon, Sabrina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cafferata, Eduardo Gustavo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Nuñez, Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Soto, Ariadna Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Sanchez Lamas, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Afonso, María Jimena. Fundación Instituto Leloir; ArgentinaFil: Aguilar Cortes, Diana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rios, Gregorio David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Maricato, Juliana T.. Universidade Federal de Sao Paulo; BrasilFil: Torres Braconi, Carla. Universidade Federal de Sao Paulo; BrasilFil: Barbosa da Silveira, Vanessa. Universidade Federal de Sao Paulo; BrasilFil: Montes de Andrade, Tatiane. Universidade Federal de Sao Paulo; BrasilFil: Carvalho de Souza Bonetti, Tatiana. Universidade Federal de Sao Paulo; BrasilFil: Ramos Janini, Luiz M.. Universidade Federal de Sao Paulo; BrasilFil: Castello Girão, Manoel J. B.. Universidade Federal de Sao Paulo; BrasilFil: Llera, Andrea Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

BF Integrase Genes of HIV-1 Circulating in São Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population

The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus proteome and the comparison of multiple isolated envelope proteins GP64.

A família Baculoviridae é um grande grupo de vírus com cerca de 700 espécies de insetos hospedeiros, com dois fenótipos: o ODV (occlusion derived virion), que faz a infecção primária do intestino médio; e o BV (budded virus), responsável pela infecção sistêmica. No Brasil, o nucleopoliedrovírus Anticarsia gemmatalis (AgMNPV) é utilizado como controle biológico da lagarta-da-soja Anticarsia gemmatalis. O genoma do AgMNPV-2D contém 152 ORFs, 26 das quais codificam proteínas estruturais. Entre elas, a glicoproteína GP64 é fundamental para infecção secundária. Este estudo visa identificar proteínas estruturais do AgMNPV-2D por duas abordagens de espectrometria de massas. Também comparar a variabilidade da gp64 de isolados geográficos por sequenciamento por Sanger e de alta cobertura. Assim, identificamos as substituições de gp64 e vimos que ela não suporta a separação geográfica dos isolados. Também identificamos 44 e 33 proteínas em ODV e BV, respectivamente. Seis novas proteínas foram identificadas no ODV e sete delas no BV. Além disso, 11 proteínas celulares foram identificadas no AgMNPV-2D, possivelmente necessárias para infecção. Este achado contribui para o entendimento da morfogênese do AgMNPV e fatores associados à multiplicação viral.Baculoviridae are arthropod-specific viruses with more than 700 host insects, which produce two phenotypes: the budded virus (BV) and, the occlusion-derived virus (ODV) for intra and across host spread, respectively. Brazil uses the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent of the velvet bean caterpillar (A. gemmatalis). The genome of the AgMNPV-2D carries 152 ORFs, 26 of which code for structural proteins. Herein, the structural proteins of AgMNPV-2D were analyzed by two mass spectrometry techniques. The additional objective was to compare the gene gp64. of different geographical populations by Sanger and next generation sequencing. This analysis allowed us to observe the substitutions of gp64 and refuted the notion of a geographical isolation of the samples. We also observed a total of 44 proteins of the ODV and 33 of the BV. Six new proteins were found in the ODV and seven in the BV. Furthermore, 11 cellular proteins were also identified, which are possibly assorted during viral morphogenesis. These findings may provide novel insights into AgMNPV biology and its host interaction, leading us to a better understanding about morphogenesis and also the associated factors of the viral multiplication

Deep phylogenetic-based clustering analysis uncovers new and shared mutations in SARS-CoV-2 variants as a result of directional and convergent evolution.

Nearly two decades after the last epidemic caused by a severe acute respiratory syndrome coronavirus (SARS-CoV), newly emerged SARS-CoV-2 quickly spread in 2020 and precipitated an ongoing global public health crisis. Both the continuous accumulation of point mutations, owed to the naturally imposed genomic plasticity of SARS-CoV-2 evolutionary processes, as well as viral spread over time, allow this RNA virus to gain new genetic identities, spawn novel variants and enhance its potential for immune evasion. Here, through an in-depth phylogenetic clustering analysis of upwards of 200,000 whole-genome sequences, we reveal the presence of previously unreported and hitherto unidentified mutations and recombination breakpoints in Variants of Concern (VOC) and Variants of Interest (VOI) from Brazil, India (Beta, Eta and Kappa) and the USA (Beta, Eta and Lambda). Additionally, we identify sites with shared mutations under directional evolution in the SARS-CoV-2 Spike-encoding protein of VOC and VOI, tracing a heretofore-undescribed correlation with viral spread in South America, India and the USA. Our evidence-based analysis provides well-supported evidence of similar pathways of evolution for such mutations in all SARS-CoV-2 variants and sub-lineages. This raises two pivotal points: (i) the co-circulation of variants and sub-lineages in close evolutionary environments, which sheds light onto their trajectories into convergent and directional evolution, and (ii) a linear perspective into the prospective vaccine efficacy against different SARS-CoV-2 strains

Phylogenetic tree using recombination-free segment.

<p>The cladogram summarizes the maximum clade credibility (MCC) tree including all subtype B integrases and the recombinant integrase sequences sharing the 500 bp subtype B segment. It shows reconstructions in “recombinant” and “non-recombinant” state transitions on a set of 5000 plausible trees by using the symmetric CTMC model for discrete character reconstruction with BEAST. Recombinant sequences are shown as thick branches, while asterisks at the tips of the MCC tree show sequences that share a breakpoint at position 4924. Branch lengths only help the visualization of transition events and are not proportional to either quantity of events or substitution per sites.</p

Integrase phylogeny.

<p>A maximum likelihood tree for 459 non-recombinant integrase sequences from Brazil (145 generated by us and 314 from other studies) inferred with GARLI v0.95. Thick branches indicate lineages known to be recombinants on other regions of their genomes. The tree was midpoint rooted, which agrees precisely with the known relationship among HIV-1 group M subtypes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034324#pone.0034324-McCutchan1" target="_blank">[34]</a>. Subtype C sequences are collapsed for clarity. This tree shows a large number of potential independent origination events of BF recombinants.</p

Recombination profiles and selection.

<p>Plot of the differences between non-synonymous (<i>dN</i>) and synonymous (<i>dS</i>) rates shown above in the mosaic maps of integrase (864 bp) with nucleotide sites numbered in relation to the HXB2. Rate values per codon site for the subtype B (blue squares) and subtype F (green circles). Blue and green striped portions indicate uncertainty on subtype provenance and usually include the breakpoints (indicated by an asterisk) estimated with jpHMM algorithm available online at the GOBICS server (<a href="http://jphmm.gobics.de/jphmm.html" target="_blank">http://jphmm.gobics.de/jphmm.html</a>). Samples 0441, 0612, 1365, and 1470 shared the same breakpoint at nucleotide 4924 that falls inside the uncertainty region from 4899 to 4977 bp but had different recombination profiles on parts of their genomes. Sample 0708 also shared a recombination spot starting at 4899 bp, but no breakpoint could be determined.</p

Variability of the conserved V3 loop tip motif in HIV-1 subtype B isolates collected from Brazilian and French patients

The diversity of the V3 loop tip motif sequences of HIV-1 subtype B was analyzed in patients from Botucatu (Brazil) and Montpellier (France). Overall, 37 tetrameric tip motifs were identified, 28 and 17 of them being recognized in Brazilian and French patients, respectively. The GPGR (P) motif was predominant in French but not in Brazilian patients (53.5% vs 31.0%), whereas the GWGR (W) motif was frequent in Brazilian patients (23.0%) and absent in French patients. Three tip motif groups were considered: P, W, and non-P non-W groups. The distribution of HIV-1 isolates into the three groups was significantly different between isolates from Botucatu and from Montpellier (P < 0.001). A higher proportion of CXCR4-using HIV-1 (X4 variants) was observed in the non-P non-W group as compared with the P group (37.5% vs 19.1%), and no X4 variant was identified in the W group (P < 0.001). The higher proportion of X4 variants in the non-P non-W group was essentially observed among the patients from Montpellier, who have been infected with HIV-1 for a longer period of time than those from Botucatu. Among patients from Montpellier, CD4+ cell counts were lower in patients belonging to the non-P non-W group than in those belonging to the P group (24 cells/µL vs 197 cells/µL; P = 0.005). Taken together, the results suggest that variability of the V3 loop tip motif may be related to HIV-1 coreceptor usage and to disease progression. However, as analyzed by a bioinformatic method, the substitution of the V3 loop tip motif of the subtype B consensus sequence with the different tip motifs identified in the present study was not sufficient to induce a change in HIV-1 coreceptor usage