Transcriptional Stochasticity as a Key Aspect of HIV-1 Latency.

This review summarizes current advances in the role of transcriptional stochasticity in HIV-1 latency, which were possible in a large part due to the development of single-cell approaches. HIV-1 transcription proceeds in bursts of RNA production, which stem from the stochastic switching of the viral promoter between ON and OFF states. This switching is caused by random binding dynamics of transcription factors and nucleosomes to the viral promoter and occurs at several time scales from minutes to hours. Transcriptional bursts are mainly controlled by the core transcription factors TBP, SP1 and NF-κb, the chromatin status of the viral promoter and RNA polymerase II pausing. In particular, spontaneous variability in the promoter chromatin creates heterogeneity in the response to activators such as TNF-α, which is then amplified by the Tat feedback loop to generate high and low viral transcriptional states. This phenomenon is likely at the basis of the partial and stochastic response of latent T cells from HIV-1 patients to latency-reversing agents, which is a barrier for the development of shock-and-kill strategies of viral eradication. A detailed understanding of the transcriptional stochasticity of HIV-1 and the possibility to precisely model this phenomenon will be important assets to develop more effective therapeutic strategies.

Anti-SARS-CoV-2 Activity of Polysaccharides Extracted from Halymenia floresii and Solieria chordalis (Rhodophyta).

Even after hundreds of clinical trials, the search for new antivirals to treat COVID-19 is still relevant. Carrageenans are seaweed sulfated polysaccharides displaying antiviral activity against a wide range of respiratory viruses. The objective of this work was to study the antiviral properties of Halymenia floresii and Solieria chordalis carrageenans against SARS-CoV-2. Six polysaccharide fractions obtained from H. floresii and S. chordalis by Enzyme-Assisted Extraction (EAE) or Hot Water Extraction (HWE) were tested. The effect of carrageenan on viral replication was assessed during infection of human airway epithelial cells with a clinical strain of SARS-CoV-2. The addition of carrageenans at different times of the infection helped to determine their mechanism of antiviral action. The four polysaccharide fractions isolated from H. floresii displayed antiviral properties while the S. chordalis fractions did not. EAE-purified fractions caused a stronger reduction in viral RNA concentration. Their antiviral action is likely related to an inhibition of the virus attachment to the cell surface. This study confirms that carrageenans could be used as first-line treatment in the respiratory mucosa to inhibit the infection and transmission of SARS-CoV-2. Low production costs, low cytotoxicity, and a broad spectrum of antiviral properties constitute the main strengths of these natural molecules.

Antiviral Effect of hBD-3 and LL-37 during Human Primary Keratinocyte Infection with West Nile Virus.

West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.

Phenol-soluble modulins α are major virulence factors of Staphylococcus aureus secretome promoting inflammatory response in human epidermis.

Staphylococcus aureus is a skin commensal microorganism commonly colonizing healthy humans. Nevertheless, S. aureus can also be responsible for cutaneous infections and contribute to flare-up of inflammatory skin diseases such as atopic dermatitis (AD), which is characterized by dysbiosis of the skin microbiota with S. aureus as the predominant species. However, the role of major virulence factors of this pathogen such as phenol-soluble modulin (PSM) toxins in epidermal inflammation remains poorly understood. Stimulation of primary human keratinocytes with sublytic concentrations of synthetic and purified PSM α3 resulted in upregulation of a large panel of pro-inflammatory chemokine and cytokine gene expression, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CCL20, IL-1α, IL-1ß, IL-6, IL-36γ and TNF-α, while inducing the release of CXCL8, CCL20, TNF-α and IL-6. In addition, using S. aureus culture supernatant from mutants deleted from genes encoding either α-type PSMs or all PSM production, PSMs were shown to be the main factors of S. aureus secretome responsible for pro-inflammatory mediator induction in human keratinocytes. On the other hand, α-type PSM-containing supernatant triggered an intense induction of pro-inflammatory mediator expression and secretion during both topical and basal layer stimulation of an ex vivo model of human skin explants, a physiologically relevant model of pluristratified epidermis. Taken together, the results of this study show that PSMs and more specifically α-type PSMs are major virulence factors of S. aureus inducing a potent inflammatory response during infection of the human epidermis and could thereby contribute to AD flare-up through exacerbation of skin inflammation.

Characterisation of Antiviral Activity of Cathelicidins from Naked Mole Rat and Python bivittatus on Human Herpes Simplex Virus 1.

Hg-CATH and Pb-CATH4 are cathelicidins from Heterocephalus glaber and Python bivittatus that have been previously identified as potent antibacterial peptides. However, their antiviral properties were not previously investigated. In this study, their activity against the herpes simplex virus (HSV)-1 was evaluated during primary human keratinocyte infection. Both of them significantly reduced HSV-1 DNA replication and production of infectious viral particles in keratinocytes at noncytotoxic concentrations, with the stronger activity of Pb-CATH4. These peptides did not show direct virucidal activity and did not exhibit significant immunomodulatory properties, except for Pb-CATH4, which exerted a moderate proinflammatory action. All in all, our results suggest that Hg-CATH and Pb-CATH4 could be potent candidates for the development of new therapies against HSV-1.

Eczema Herpeticum: Clinical and Pathophysiological Aspects.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the world. AD is a complex pathology mainly characterized by an impaired skin barrier, immune response dysfunction, and unbalanced skin microbiota. Moreover, AD patients exhibit an increased risk of developing bacterial and viral infections. One of the most current, and potentially life-threatening, viral infection is caused by herpes simplex virus (HSV), which occurs in about 3% of AD patients under the name of eczema herpeticum (EH). Following a first part dedicated to the clinical features, virological diagnosis, and current treatments of EH, this review will focus on the description of the pathophysiology and, more particularly, the presently known predisposing factors to herpetic complications in AD patients. These factors include those related to impairment of the skin barrier such as deficit in filaggrin and anomalies in tight and adherens junctions. In addition, low production of the antimicrobial peptides cathelicidin LL-37 and human ß-defensins; overexpression of cytokines such as interleukin (IL)-4, IL-13, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP); or downregulation of type I to III interferons as well as defect in functions of immune cells such as dendritic, natural killer, and regulatory T cells have been involved. Otherwise, genetic polymorphisms and AD topical calcineurin inhibitor treatments have been associated with an increased risk of EH. Finally, dysbiosis of skin microbiota characterized in AD patients by Staphylococcus aureus colonization and toxin secretion, such as α-toxin, has been described as promoting HSV replication and could therefore contribute to EH.

Comparison of Anti-Viral Activity of Frog Skin Anti-Microbial Peptides Temporin-Sha and [K³]SHa to LL-37 and Temporin-Tb against Herpes Simplex Virus Type 1.

Temporins are anti-microbial peptides synthesized in the skin of frogs of the Ranidae family. The few studies to date that have examined their anti-viral properties have shown that they have potential as anti-viral therapies. In this work, we evaluated the anti-herpes simplex virus type 1 (HSV-1) activity of the temporin-SHa (SHa) and its synthetic analog [K³]SHa. Human cathelicidin LL-37 and temporin-Tb (Tb), previously demonstrated to have anti-HSV-1 properties, were used as positive controls. We observed that SHa and [K³]SHa significantly inhibit HSV-1 replication in human primary keratinocytes when used at micromolar concentrations. This anti-viral activity was equivalent to that of Tb, but lower than that of LL-37. Transcriptomic analyses revealed that SHa did not act through the modulation of the cell innate immune response, but rather, displayed virucidal properties by reducing infectious titer of HSV-1 in suspension. In contrast, pre-incubation of the virus with LL-37 suggests that this peptide does not act directly on the viral particle at non-cytotoxic concentrations tested. The anti-HSV-1 activity of LL-37 appears to be due to the potentiation of cellular anti-viral defenses through the induction of interferon stimulated gene expression in infected primary keratinocytes. This study demonstrated that SHa and [K³]SHa, in addition to their previously reported antibacterial and antiparasitic activities, are direct-acting anti-HSV-1 peptides. Importantly, this study extends the little studied anti-viral attributes of frog temporins and offers perspectives for the development of new anti-HSV-1 therapies.

Innate Immune Response of Primary Human Keratinocytes to West Nile Virus Infection and Its Modulation by Mosquito Saliva.

West Nile Virus (WNV) is a flavivirus involved in many human infections worldwide. This arthropod-borne virus is directly co-inoculated with mosquito saliva through the epidermis and the dermis during blood meal. WNV starts replicating in the skin before migrating to the draining lymph node, leading to widespread viremia and in some cases to neurological symptoms. Skin is a complex organ composed of different cell types that together perform essential functions such as pathogen sensing, barrier maintenance and immunity. Keratinocytes, which represent 90% of the cells of the epidermis, are the organism's first line of defense, initiating innate immune response by recognizing pathogens through their pattern recognition receptors. Although WNV was previously known to replicate in human primary keratinocytes, the induced inflammatory response remains unknown. The aim of this study was first to characterize the inflammatory response of human primary keratinocytes to WNV infection and then, to assess the potential role of co-inoculated mosquito saliva on the keratinocyte immune response and viral replication. A type I and III interferon inflammatory response associated with an increase of IRF7 but not IRF3 mRNA expression, and dependent on infectious dose, was observed during keratinocyte infection with WNV. Expression of several interferon-stimulated gene mRNA was also increased at 24 h post-infection (p.i.); they included CXCL10 and interferon-induced proteins with tetratricopeptide repeats (IFIT)-2 sustained up until 48 h p.i. Moreover, WNV infection of keratinocyte resulted in a significant increase of pro-inflammatory cytokines (TNFα, IL-6) and various chemokines (CXCL1, CXCL2, CXCL8 and CCL20) expression. The addition of Aedes aegypti or Culex quinquefasciatus mosquito saliva, two vectors of WNV infection, to infected keratinocytes led to a decrease of inflammatory response at 24 h p.i. However, only Ae. Aegypti saliva adjunction induced modulation of viral replication. In conclusion, this work describes for the first time the inflammatory response of human primary keratinocytes to WNV infection and its modulation in presence of vector mosquito saliva. The effects of mosquito saliva assessed in this work could be involved in the early steps of WNV replication in skin promoting viral spread through the body.