Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity.

Persistent HIV-1 transcription during ART: time to reassess its significance?

Purpose Of Review: Despite suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and reignite viral replication if therapy is interrupted. Persistence of the viral reservoir in people with HIV-1 (PWH) is the main obstacle to an HIV-1 cure. The reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals.

AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells.

CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.

Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

Background: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates.

Methods: Commercial CA-p24 antibodies were used as capture and detection antibodies.

Recent Advances Using Genetic Therapies Against Infectious Diseases and for Vaccination.

The development of prophylatic or therapeutic medicines for infectious diseases is one of the priorities for health organizations worldwide. Innovative solutions are required to achieve effective, safe, and accessible treatments for most if not all infectious diseases, particularly those that are chronic in nature or that emerge unexpectedly over time. Genetic technologies offer versatile possibilities to design therapies against pathogens.

CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures.

Background: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a.

Methods: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs.

HIV-1 3'-Polypurine Tract Mutations Confer Dolutegravir Resistance by Switching to an Integration-Independent Replication Mechanism via 1-LTR Circles.

Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII.

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2.

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence.

HIV persistence: silence or resistance?

Despite decades of suppressive antiretroviral therapy, human immunodeficiency virus (HIV) reservoirs in infected individuals persist and fuel viral rebound once therapy is interrupted. The persistence of viral reservoirs is the main obstacle to achieving HIV eradication or a long-term remission. The last decade has seen a profound change in our understanding of the mechanisms behind HIV persistence, which appears to be much more complex than originally assumed.

HIV co-opts a cellular antiviral mechanism, activation of stress kinase PKR by its RNA, to enable splicing of rev/tat mRNA.

Background: Activation of RNA-dependent stress kinase PKR, especially by viral double-stranded RNA, induces eukaryotic initiation factor 2 α-chain (eIF2α) phosphorylation, attenuating thereby translation. We report that this RNA-mediated negative control mechanism, considered a cornerstone of the cell's antiviral response, positively regulates splicing of a viral mRNA.

Results: Excision of the large human immunodeficiency virus (HIV) rev/tat intron depends strictly on activation of PKR by the viral RNA and on eIF2α phosphorylation.

A CRISPR-Cas Cure for HIV/AIDS.

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy.

Silent codon positions in the A-rich HIV RNA genome that do not easily become A: Restrictions imposed by the RNA sequence and structure.

There is a strong evolutionary tendency of the human immunodeficiency virus (HIV) to accumulate A nucleotides in its RNA genome, resulting in a mere 40 per cent A count. This A bias is especially dominant for the so-called silent codon positions where any nucleotide can be present without changing the encoded protein. However, particular silent codon positions in HIV RNA refrain from becoming A, which became apparent upon genome analysis of many virus isolates.

Towards a molecular profile of antiretroviral therapy-free HIV remission.

Purpose Of Review: To summarize the current status and highlight recent findings on predictive biomarkers for posttreatment HIV control (PTC) and virological remission. While historically, many studies focused on virological markers, there is an increasing tendency to enter immune and metabolic factors into the equation.

Recent Findings: On the virological side, several groups reported that cell-associated HIV RNA could predict time to viral rebound.

Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.

Background: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC).

Phylogenetic estimation of the viral fitness landscape of HIV-1 set-point viral load.

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 10 and 10 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences.

A combinatorial CRISPR-Cas12a attack on HIV DNA.

CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack.

SARS-CoV-2 Evolution: On the Sudden Appearance of the Omicron Variant.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant.

In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack.

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy.

A highly virulent variant of HIV-1 circulating in the Netherlands.

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.