Selective clonal persistence of human retroviruses in vivo: radial chromatin organization, integration site and host transcription

The human retroviruses HTLV-1 and HIV-1 persist in vivo, despite the host immune response and antiretroviral therapy, as a reservoir of latently infected T-cell clones. It is poorly understood what determines which clones survive in the reservoir and which are lost. We compared >160,000 HTLV-1 integration sites from T-cells isolated ex vivo from naturally-infected subjects with >230,000 integration sites from in vitro infection, to identify the genomic features that determine selective clonal survival. Three factors explained >40% of the observed variance in clone survival of HTLV-1 in vivo: the radial intranuclear position of the provirus, its absolute genomic distance from the centromere, and the intensity of host genome transcription flanking the provirus. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for transcriptionally repressive nuclear compartments favours clonal persistence of human retroviruses in vivo.

Non-Coding Genome, Transcription Factors, and Sex Determination

In vertebrates, gonadal sex determination is the process by which transcription factors drive the choice between the testicular and ovarian identity of undifferentiated somatic progenitors through activation of 2 different transcriptional programs. Studies in animal models suggest that sex determination always involves sex-specific transcription factors that activate or repress sex-specific genes. These transcription factors control their target genes by recognizing their regulatory elements in the non-coding genome and their binding motifs within their DNA sequence. In the last 20 years, the development of genomic approaches that allow identifying all the genomic targets of a transcription factor in eukaryotic cells gave the opportunity to globally understand the function of the nuclear proteins that control complex genetic programs. Here, the major transcription factors involved in male and female vertebrate sex determination and the genomic profiling data of mouse gonads that contributed to deciphering their transcriptional regulation role will be reviewed.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, RNA virus in the family Pneumoviridae and genus Orthopneumoviridae that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all serine S and T residues to alanine (A) and first analyzed their effect on genome transcription and replication using a minigenome system. We found that the mutation of eight residues resulted in significantly reduced minigenome activity compared to wild-type P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (T151A, S156A, T160A, and S23A), suggesting RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four rescued, rRSV-T160A caused a minor growth defect compared to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication, and S156 plays a critical role in viral RNA synthesis. Importance RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylate these residues can lead to kinase inhibitors and anti-viral drugs for this important human pathogen.

Comparison of Zaire and Bundibugyo ebolavirus polymerase complexes and susceptibility to antivirals through a newly developed Bundibugyo minigenome system

Members of the genus Ebolavirus cause lethal disease in humans with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small molecule inhibtors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3’-untranslated region) and replication (5’-untranslated region). Varying levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3’-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, post-transfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV compared to EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex may be a viable target for therapeutics. Importance Ebolavirus disease is a viral infection and is fatal in 25-90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species but there is still limited information regarding the viral mechanism for these differences. Previous studies suggest that BDBV replicates slower than EBOV but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system which can be used in a biosafety level (BSL) 2 laboartory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

Support Vector Machine as a Supervised Learning for the Prioritization of Novel Potential SARS-CoV-2 Main Protease Inhibitors

In the last year, the COVID-19 pandemic has highly affected the lifestyle of the world population, encouraging the scientific community towards a great effort on studying the infection molecular mechanisms. Several vaccine formulations are nowadays available and helping to reach immunity. Nevertheless, there is a growing interest towards the development of novel anti-covid drugs. In this scenario, the main protease (Mpro) represents an appealing target, being the enzyme responsible for the cleavage of polypeptides during the viral genome transcription. With the aim of sharing new insights for the design of novel Mpro inhibitors, our research group developed a machine learning approach using the support vector machine (SVM) classification. Starting from a dataset of two million commercially available compounds, the model was able to classify two hundred novel chemo-types as potentially active against the viral protease. The compounds labelled as actives by SVM were next evaluated through consensus docking studies on two PDB structures and their binding mode was compared to well-known protease inhibitors. The best five compounds selected by consensus docking were then submitted to molecular dynamics to deepen binding interactions stability. Of note, the compounds selected via SVM retrieved all the most important interactions known in the literature.

Transcriptional Responses of Sclerotinia sclerotiorum to the Infection by SsHADV-1

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

RUNX1-mediated alphaherpesvirus-host trans-species chromatin interaction promotes viral transcription

Like most DNA viruses, herpesviruses precisely deliver their genomes into the sophisticatedly organized nuclei of the infected host cells to initiate subsequent transcription and replication. However, it remains elusive how the viral genome specifically interacts with the host genome and hijacks host transcription machinery. Using pseudorabies virus (PRV) as model virus, we performed chromosome conformation capture assays to demonstrate a genome-wide specific trans-species chromatin interaction between the virus and host. Our data show that the PRV genome is delivered by the host DNA binding protein RUNX1 into the open chromatin and active transcription zone. This facilitates virus hijacking host RNAPII to efficiently transcribe viral genes, which is significantly inhibited by either a RUNX1 inhibitor or RNA interference. Together, these findings provide insights into the chromatin interaction between viral and host genomes and identify new areas of research to advance the understanding of herpesvirus genome transcription.

Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures

The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as “restriction factors”) target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

The Polarity of an Amino Acid at Position 1891 of Severe Fever with Thrombocytopenia Syndrome Virus L Protein Is Critical for the Polymerase Activity

Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.

The RNA Architecture of the SARS-CoV-2 3′-Untranslated Region

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.