Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein

Aims: To study effects on cellular innate immune responses to novel genes ORF8 and ORF10, and the more conserved Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, either alone, or in combination with cannabidiol (CBD). Main Methods: HEK293 cells were transfected with a control plasmid, or plasmids expressing ORF8, ORF10, or M protein, and assayed for cell number and markers of apoptosis at 24 h, and expression of interferon and interferon-stimulated genes at 14 h. Key findings: A significant reduction in cell number, and increase in early and late apoptosis, was found after 24 h in cells where expression of viral genes was combined with 1-2 μM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. CBD (2 μM) augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2'-5'-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL, in cells expressing ORF8, ORF10, and M protein. CBD also augmented expression of these genes in control cells not expressing viral genes, without enhancing apoptosis. Significance: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but suggest an augmented innate anti-viral response to these genes in the presence of CBD. Furthermore, our results indicate that CBD may prime components of the innate immune system, increasing readiness to respond to viral infection without activating apoptosis, and therefore could be studied for potential in prophylaxis.

The first report of porcine parvovirus 7 (PPV7) in Colombia demonstrates the presence of variants associated with modifications at the level of the VP2-capsid protein

There are a wide variety of porcine parvoviruses (PPVs) referred to as PPV1 to PPV7. The latter was discovered in 2016 and later reported in some countries in America, Asia, and Europe. PPV7 as a pathogenic agent or coinfection with other pathogens causing disease has not yet been determined. In the present study, we report the identification of PPV7 for the first time in Colombia, where it was found retrospectively since 2015 in 40% of the provinces that make up the country (13/32), and the virus was ratified for 2018 in 4/5 provinces evaluated. Additionally, partial sequencing (nucleotides 380 to 4000) was performed of four Colombian strains completely covering the VP2 and NS1 viral genes. A sequence identity greater than 99% was found when comparing them with reference strains from the USA and China. In three of the four Colombian strains, an insertion of 15 nucleotides (five amino acids) was found in the PPV7-VP2 capsid protein (540–5554 nt; 180–184 aa). Based on this insertion, the VP2 phylogenetic analysis exhibited two well-differentiated evolutionarily related groups. To evaluate the impact of this insertion on the structure of the PPV7-VP2 capsid protein, the secondary structure of two different Colombian strains was predicted, and it was determined that the insertion is located in the coil region and not involved in significant changes in the structure of the protein. The 3D structure of the PPV7-VP2 capsid protein was determined by threading and homology modeling, and it was shown that the insertion did not imply a change in the shape of the protein. Additionally, it was determined that the insertion is not involved in suppressing a potential B cell epitope, although the increase in length of the epitope could affect the interaction with molecules that allow a specific immune response.

High content screening and computational prediction reveal viral genes that suppress innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-kB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

SummaryThe highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two- photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons’ visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals.

Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Development and Adoption of Genetically Engineered Plants for Virus Resistance: Advances, Opportunities and Challenges

Plant viruses cause yield losses to crops of agronomic and economic significance and are a challenge to the achievement of global food security. Although conventional plant breeding has played an important role in managing plant viral diseases, it will unlikely meet the challenges posed by the frequent emergence of novel and more virulent viral species or viral strains. Hence there is an urgent need to seek alternative strategies of virus control that can be more readily deployed to contain viral diseases. The discovery in the late 1980s that viral genes can be introduced into plants to engineer resistance to the cognate virus provided a new avenue for virus disease control. Subsequent advances in genomics and biotechnology have led to the refinement and expansion of genetic engineering (GE) strategies in crop improvement. Importantly, many of the drawbacks of conventional breeding, such as long lead times, inability or difficulty to cross fertilize, loss of desirable plant traits, are overcome by GE. Unfortunately, public skepticism towards genetically modified (GM) crops and other factors have dampened the early promise of GE efforts. These concerns are principally about the possible negative effects of transgenes to humans and animals, as well as to the environment. However, with regards to engineering for virus resistance, these risks are overstated given that most virus resistance engineering strategies involve transfer of viral genes or genomic segments to plants. These viral genomes are found in infected plant cells and have not been associated with any adverse effects in humans or animals. Thus, integrating antiviral genes of virus origin into plant genomes is hardly unnatural as suggested by GM crop skeptics. Moreover, advances in deep sequencing have resulted in the sequencing of large numbers of plant genomes and the revelation of widespread endogenization of viral genomes into plant genomes. This has raised the possibility that viral genome endogenization is part of an antiviral defense mechanism deployed by the plant during its evolutionary past. Thus, GM crops engineered for viral resistance would likely be acceptable to the public if regulatory policies were product-based (the North America regulatory model), as opposed to process-based. This review discusses some of the benefits to be gained from adopting GE for virus resistance, as well as the challenges that must be overcome to leverage this technology. Furthermore, regulatory policies impacting virus-resistant GM crops and some success cases of virus-resistant GM crops approved so far for cultivation are discussed.

Speckles and paraspeckles coordinate to regulate HSV-1 genes transcription

Numbers of nuclear speckles and paraspeckles components have been demonstrated to regulate herpes simplex virus 1 (HSV-1) replication. However, how HSV-1 infection affects the two nuclear bodies, and whether this influence facilitates the expression of viral genes, remains elusive. In the current study, we found that HSV-1 infection leads to a redistribution of speckles and paraspeckles components. Serine/arginine-rich splicing factor 2 (SRSF2), the core component of speckles, was associated with multiple paraspeckles components, including nuclear paraspeckles assembly transcript 1 (NEAT1), PSPC1, and P54nrb, in HSV-1 infected cells. This association coordinates the transcription of viral genes by binding to the promoters of these genes. By association with the enhancer of zeste homolog 2 (EZH2) and P300/CBP complex, NEAT1 and SRSF2 influenced the histone modifications located near viral genes. This study elucidates the interplay between speckles and paraspeckles following HSV-1 infection and provides insight into the mechanisms by which HSV-1 utilizes host cellular nuclear bodies to facilitate its life cycle.

Tracking the Transcription Kinetic of SARS-CoV-2 in Human Cells by Reverse Transcription-Droplet Digital PCR

Viral transcription is an essential step of SARS-CoV-2 infection after invasion into the target cells. Antiviral drugs such as remdesivir, which is used to treat COVID-19 patients, targets the viral RNA synthesis. Understanding the mechanism of viral transcription may help to develop new therapeutic treatment by perturbing virus replication. In this study, we established 28 ddPCR assays and designed specific primers/probe sets to detect the RNA levels of 15 NSP, 9 ORF, and 4 structural genes of SARS-CoV-2. The transcriptional kinetics of these viral genes were determined longitudinally from the beginning of infection to 12 hours postinfection in Caco-2 cells. We found that SARS-CoV-2 takes around 6 hours to hijack the cells before the initiation of viral transcription process in human cells. Our results may contribute to a deeper understanding of the mechanisms of SARS-CoV-2 infection.

FAST Proteins: Development and Use of Reverse Genetics Systems for Reoviridae Viruses

Reverse genetics systems for viruses, the technology used to generate gene-engineered recombinant viruses from artificial genes, enable the study of the roles of the individual nucleotides and amino acids of viral genes and proteins in infectivity, replication, and pathogenicity. The successful development of a reverse genetics system for poliovirus in 1981 accelerated the establishment of protocols for other RNA viruses important for human health. Despite multiple efforts, rotavirus (RV), which causes severe gastroenteritis in infants, was refractory to reverse genetics analysis, and the first complete reverse genetics system for RV was established in 2017. This novel technique involves use of the fusogenic protein FAST (fusion-associated small transmembrane) derived from the bat-borne Nelson Bay orthoreovirus, which induces massive syncytium formation. Co-transfection of a FAST-expressing plasmid with complementary DNAs encoding RV genes enables rescue of recombinant RV. This review focuses on methodological insights into the reverse genetics system for RV and discusses applications and potential improvements to this system.