Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells

Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components

The role of zinc in antiviral immunity

Zinc is an essential trace element that is crucial for growth, development, and the maintenance of immune function. Its influence reaches all organs and cell types, representing an integral component of approximately 10% of the human proteome, and encompassing hundreds of key enzymes and transcription factors. Zinc deficiency is strikingly common, affecting up to a quarter of the population in developing countries, but also affecting distinct populations in the developed world as a result of lifestyle, age, and disease-mediated factors. Consequently, zinc status is a critical factor that can influence antiviral immunity, particularly as zinc-deficient populations are often most at risk of acquiring viral infections such as HIV or hepatitis C virus. This review summarizes current basic science and clinical evidence examining zinc as a direct antiviral, as well as a stimulant of antiviral immunity. An abundance of evidence has accumulated over the past 50 y to demonstrate the antiviral activity of zinc against a variety of viruses, and via numerous mechanisms. The therapeutic use of zinc for viral infections such as herpes simplex virus and the common cold has stemmed from these findings; however, there remains much to be learned regarding the antiviral mechanisms and clinical benefit of zinc supplementation as a preventative and therapeutic treatment for viral infections

Controlling HIV-1: Non-coding RNA gene therapy approaches to a functional cure

The current treatment strategy for HIV-1 involves prolonged and intensive combined antiretroviral therapy (cART), which successfully suppresses plasma viremia. It has transformed HIV-1 infection into a chronic disease. However, despite the success of cART, a latent form of HIV-1 infection persists as integrated provirus in resting memory CD4+ T cells. Virus can reactivate from this reservoir upon cessation of treatment and hence HIV requires lifelong therapy. The reservoir represents a major barrier to eradication. Understanding molecular mechanisms regulating HIV-1 transcription and latency are crucial to developing alternate treatment strategies which impact upon the reservoir and provide a path towards a functional cure in which there is no detectable viremia in the absence of cART. Numerous reports have suggested ncRNAs are involved in regulating viral transcription and latency. This review will discuss the latest developments in ncRNAs, specifically short interfering (si)RNA and short hairpin (sh)RNA, targeting molecular mechanisms of HIV-1 transcription, which may represent potential future therapeutics. It will also briefly address animal models available for testing potential therapeutics and current gene therapy clinical trials

Data from: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

Lambda interferons (IFNL, IFN-λ) are pro-inflammatory cytokines important in acute and chronic viral infection. Single-nucleotide polymorphisms rs12979860 and rs8099917 within the IFNL gene locus predict hepatitis C virus (HCV) clearance, as well as inflammation and fibrosis progression in viral and non-viral liver disease. The underlying mechanism, however, is not defined. Here we show that the rs12979860 CC genotype correlates with increased hepatic metallothionein expression through increased systemic zinc levels. Zinc interferes with IFN-λ3 binding to IFNL receptor 1 (IFNLR1), resulting in decreased antiviral activity and increased viral replication (HCV, influenza) in vitro. HCV patients with high zinc levels have low hepatocyte antiviral and inflammatory gene expression and high viral loads, confirming the inhibitory role of zinc in vivo. We provide the first evidence that zinc can act as a potent and specific inhibitor of IFN-λ3 signalling and highlight its potential as a target of therapeutic intervention for IFN-λ3-mediated chronic disease

Zinc is a potent and specific inhibitor of IFN-λ3 signalling

Lambda interferons (IFNL, IFN-λ) are pro-inflammatory cytokines important in acute and chronic viral infection. Single-nucleotide polymorphisms rs12979860 and rs8099917 within the IFNL gene locus predict hepatitis C virus (HCV) clearance, as well as inflammation and fibrosis progression in viral and non-viral liver disease. The underlying mechanism, however, is not defined. Here we show that the rs12979860 CC genotype correlates with increased hepatic metallothionein expression through increased systemic zinc levels. Zinc interferes with IFN-λ3 binding to IFNL receptor 1 (IFNLR1), resulting in decreased antiviral activity and increased viral replication (HCV, influenza) in vitro. HCV patients with high zinc levels have low hepatocyte antiviral and inflammatory gene expression and high viral loads, confirming the inhibitory role of zinc in vivo. We provide the first evidence that zinc can act as a potent and specific inhibitor of IFN-λ3 signalling and highlight its potential as a target of therapeutic intervention for IFN-λ3-mediated chronic disease

Nanoscale probing and imaging of HIV-1 RNA in cells with a chimeric LNA-DNA sensor

Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network

Statement in Support of: “Virology under the Microscope—a Call for Rational Discourse”

[Extract] We, members of the Australasian Virology Society, agree with and support the statement entitled “Virology under the Microscope—a Call for Rational Discourse” (1). Like virologists everywhere, we have worked with scientist and clinician colleagues worldwide to develop knowledge, tests, and interventions which collectively have reduced the number of deaths due to COVID-19 and curtailed its economic impact. Such work adds to the extraordinary achievements resulting from virology research that have delivered vaccines and/or antivirals against a long list of diseases and global scourges, including AIDS, smallpox, and polio (1). We believe the question of the origin of SARS-CoV-2 should be approached with an open mind and in consideration of the best scientific evidence available. We concur with the view that the zoonosis hypothesis has the strongest supporting evidence (2–4), and this is a scenario that has been observed repeatedly in the past (5), including in Australia (6). Recent data strongly support the zoonosis hypothesis (7). We share the concern that emotive and fear-based dialogues in this area add to public confusion and can lead to ill-informed condemnation of virology research