89 research outputs found
The silence within: a conserved intragenic silencing element governs HTLV-1 expression via host RUNX1 complex binding
No full textA defining feature of Human T-cell leukaemia virus type 1 (HTLV-1) infection is the establishment of reversible latency that can persist for many years. This latency enables the expansion of infected CD4+ T cells, ultimately contributing to adult T-cell leukaemia (ATL) and inflammatory disease. Sugata et al. identify a viral negative regulatory element within the HTLV-1 proviral genome that governs transcriptional latency. This intragenic silencing element contains binding sites for the master haematopoietic transcription factor, RUNX1. RUNX1 complex binding represses viral expression, thereby reducing viral production, antigen presentation, and susceptibility to cytotoxic T lymphocyte responses. The intragenic silencing element described in their study is unique to HTLV-1 and represents a novel strategy by which the virus achieves lifelong persistence in the host.10.1038/s44298-025-00136-
Protocol for production and expression of chimeric bovine-human monoclonal antibodies
No full textWe describe herein a protocol for production of chimeric bovine-human monoclonal antibodies (mAbs) from vaccinated cows. The genes of HIV-1-specific single B cells are amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned into human expression vectors, and expressed in human cell lines. This protocol provides an efficient step-by-step methodology to produce HIV-1 chimeric mAbs and could be widely adapted for other antigens. For complete details on the use and execution of this protocol, please refer to Heydarchi et al. (2022).(1
The human non‐lineage antigen CD46 (HuLy‐m5) and primate retroviral gp70 molecules share protein‐defined antigenic determinants
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A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein
No full textThe Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9’s C-terminal GxxxG-helix. In enzymatic assays, oridonin’s binding to Nsp9 reduces its potential to act as substrate for Nsp12’s Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication
Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice
No full textDuring attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons revealed that the genome sequence of the new virus was 91-93 % identical to those of known murine hepatitis viruses (MHVs). The predicted open reading frame from the nucleotide sequence encoded all known proteins of MHVs. Analysis at the protein level showed that the virus was closely related to the highly virulent MHV-JHM strain. The virus strain was named MHV-MI. No type D retroviruses were found. Degenerate PCR targeting of type D retrovirus and 5'-RACE targeting of other types of retroviruses confirmed the absence of any retroviral association with the LCH xenografted SCID mice. © 2015, The Author(s)
The Efficacy of Common Household Cleaning Agents for SARS-CoV-2 Infection Control
No full textThe COVID-19 pandemic caused by SARS-CoV-2 is having devastating effects on a global scale. Since common household disinfectants are often used to minimise the risk of infection in the home and work environment, we investigated the ability of some of these products to inactivate the virus. We tested generic brands of vinegar, bleach, and dishwashing detergent, as well as laboratory-grade acetic acid, sodium hypochlorite, and ethanol. Assays were conducted at room temperature (18-20 °C, 40% relative humidity), and two time points were used to reflect a quick wipe (30 s) and a brief soak (5 min). Vinegar, and its active ingredient, acetic acid, were completely ineffective at virus inactivation even when exposed to the virus at 90% v/v (a final concentration equivalent to 3.6% v/v acetic acid). In contrast, ethanol was capable of inactivating the virus at dilutions as low as 40% v/v. Dishwashing detergent effectively rendered SARS-CoV-2 inactive when diluted 100-fold (1% v/v). Bleach was found to be fully effective against SARS-CoV-2 at 0.21 g/L sodium hypochlorite after a 30 s exposure (1/200 dilution of commercial product). Given reports of infectious virus recovered from the surface of frozen packaging, we tested the persistence of infectiousness after multiple freeze-thaw cycles and found no change in infectious SARS-CoV-2 titre after seven freeze-thaw cycles. These results should help inform readers of how to effectively disinfect surfaces and objects that have potentially been contaminated with SARS-CoV-2 using common household chemicals
Evolutionary rate of SARS-CoV-2 increases during zoonotic infection of farmed mink
No full textTo investigate genetic signatures of adaptation to the mink host, we characterised the evolutionary rate heterogeneity in mink-associated severe acute respiratory syndrome coronaviruses (SARS-CoV-2). In 2020, the first detected anthropozoonotic spillover event of SARS-CoV-2 occurred in mink farms throughout Europe and North America. Both spill-back of mink-associated lineages into the human population and the spread into the surrounding wildlife were reported, highlighting the potential formation of a zoonotic reservoir. Our findings suggest that the evolutionary rate of SARS-CoV-2 underwent an episodic increase upon introduction into the mink host before returning to the normal range observed in humans. Furthermore, SARS-CoV-2 lineages could have circulated in the mink population for a month before detection, and during this period, evolutionary rate estimates were between 3 × 10-3 and 1.05 × 10-2 (95 per cent HPD, with a mean rate of 6.59 × 10-3) a four- to thirteen-fold increase compared to that in humans. As there is evidence for unique mutational patterns within mink-associated lineages, we explored the emergence of four mink-specific Spike protein amino acid substitutions Y453F, S1147L, F486L, and Q314K. We found that mutation Y453F emerged early in multiple mink outbreaks and that mutations F486L and Q314K may co-occur. We suggest that SARS-CoV-2 undergoes a brief, but considerable, increase in evolutionary rate in response to greater selective pressures during species jumps, which may lead to the occurrence of mink-specific mutations. These findings emphasise the necessity of ongoing surveillance of zoonotic SARS-CoV-2 infections in the future
Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor
No full textThe mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron
The RNA-Binding Proteins SRP14 and HMGB3 Control HIV-1 Tat mRNA Processing and Translation During HIV-1 Latency
No full textHIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4+ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4+ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets
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