Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

Update of Natural Products and Their Derivatives Targeting Epstein-Barr Infection.

Epstein-Barr (EBV) is a human γ-herpesvirus that undergoes both a productive (lytic) cycle and a non-productive (latent) phase. The virus establishes enduring latent infection in B lymphocytes and productive infection in the oral mucosal epithelium. Like other herpesviruses, EBV expresses its genes in a coordinated pattern during acute infection. Unlike others, it replicates its DNA during latency to maintain the viral genome in an expanding pool of B lymphocytes, which are stimulated to divide upon infection. The reactivation from the latent state is associated with a productive gene expression pattern mediated by virus-encoded transcriptional activators BZLF-1 and BRLF-1. EBV is a highly transforming virus that contributes to the development of human lymphomas. Though viral vectors and mRNA platforms have been used to develop an EBV prophylactic vaccine, currently, there are no vaccines or antiviral drugs for the prophylaxis or treatment of EBV infection and EBV-associated cancers. Natural products and bioactive compounds are widely studied for their antiviral potential and capability to modulate intracellular signaling pathways. This review was intended to collect information on plant-derived products showing their antiviral activity against EBV and evaluate their feasibility as an alternative or adjuvant therapy against EBV infections and correlated oncogenesis in humans.

HPV38 impairs UV-induced transcriptional activation of the IL-18 pro-inflammatory cytokine.

IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

Lyon IARC Polyomavirus Displays Transforming Activities in Primary Human Cells.

Several studies reported the presence of a recently discovered polyomavirus (PyV), Lyon IARC PyV (LIPyV), in human and domestic animal specimens. LIPyV has some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV (MCPyV), respectively. In this study, we demonstrate that LIPyV early proteins immortalize human foreskin keratinocytes. LIPyV LT binds pRb, accordingly cell cycle checkpoints are altered in primary human fibroblasts and keratinocytes expressing LIPyV early genes. Mutation of the pRb binding site in LT strongly affected the ability of LIPyV ER to induced HFK immortalization. LIPyV LT also binds p53 and alters p53 functions activated by cellular stresses. Finally, LIPyV early proteins activate telomerase reverse transcriptase (hTERT) gene expression, via accumulation of the Sp1 transcription factor. Sp1 recruitment to the hTERT promoter is controlled by its phosphorylation, which is mediated by ERK1 and CDK2. Together, these data highlight the transforming properties of LIPyV in in vitro experimental models, supporting its possible oncogenic nature. IMPORTANCE Lyon IARC PyV is a recently discovered polyomavirus that shows some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV, respectively. Here, we show the capability of LIPyV to efficiently promote cellular transformation of primary human cells, suggesting a possible oncogenic role of this virus in domestic animals and/or humans. Our study identified a novel virus-mediated mechanism of activation of telomerase reverse transcriptase gene expression, via accumulation of the Sp1 transcription factor. In addition, because the persistence of infection is a key event in virus-mediated carcinogenesis, it will be important to determine whether LIPyV can deregulate immune-related pathways, similarly to the well-established oncogenic viruses.

Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression.

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.

Transforming Properties of Beta-3 Human Papillomavirus E6 and E7 Proteins.

The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.

Merkel Cell Polyomavirus Downregulates N-myc Downstream-Regulated Gene 1, Leading to Cellular Proliferation and Migration.

Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.

Cross-talk of cutaneous beta human papillomaviruses and the immune system: determinants of disease penetrance.

Human papillomaviruses (HPVs) infect the epithelia of skin or mucosa, where they can induce hyperproliferative lesions. More than 220 different HPV types have been characterized and classified into five different genera. Mucosal high-risk HPVs are causative for cancers of the anogenital region and oropharynx. Clinical data from patients with the rare genetic disorder epidermodysplasia verruciformis (EV) indicate that genus beta-HPVs cooperate with ultraviolet (UV) radiation in the development of cutaneous squamous cell carcinoma. In addition, epidemiological and biological findings indicate that beta-HPV types play a role in UV-mediated skin carcinogenesis also in non-EV individuals. However, the mechanisms used by these cutaneous viruses to promote epithelial carcinogenesis differ significantly from those of mucosal HPVs. Recent studies point to a delicate cross-talk of beta-HPVs with the cell-autonomous immunity of the host keratinocytes and the local immune microenvironment that eventually determines the fate of cutaneous HPV infection and the penetrance of disease. This review gives an overview of the critical interactions of genus beta-HPVs with the local immune system that allow the virus to complete its life cycle, to escape from extrinsic immunity, and eventually to cause chronic inflammation contributing to skin carcinogenesis. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

HSV-1\EGFP stimulates miR-146a expression in a NF-κB-dependent manner in monocytic THP-1 cells.

The nuclear factor κB (NF-κB) pathway plays a key role in innate and adaptive immunity, cell proliferation and survival, inflammation and tumors development. MiR-146a is an immune system regulator that has anti-inflammatory function in multiple cell types and conditions. Here we demonstrate activation of canonical NF-κB pathway in monocytic cells upon HSV-1 replication. By constructing and using a recombinant HSV-1\EGFP virus, we monitored the capability of the virus to recruit NF-κB and we report that the phosphorylation of p65 protein correlates with an active virus replication at single-cell level. In addition, we found that upregulation of miR-146a during viral replication is strictly dependent on NF-κB activation and correlates with tight control of the interleukin-1 receptor-associate kinase 1 (IRAK1). Accordingly, THP-1 DN IκBα cells, expressing a dominant negative mIκBα, did not show upregulation of miR-146a upon HSV-1 infection. Our data suggest that the expression of miRNA-146a modulates NF-κB activation through targeting IRAK1 during HSV-1 replication in THP-1 cells.

The Abrogation of Phosphorylation Plays a Relevant Role in the CCR5 Signalosome Formation with Natural Antibodies to CCR5.

The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B ß-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that ß-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that ß-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV.

The Role of Natural Antibodies to CC Chemokine Receptor 5 in HIV Infection.

The CC chemokine receptor 5 (CCR5) is responsible for immune and inflammatory responses by mediation of chemotactic activity in leukocytes, although it is expressed on different cell types. It has been shown to act as co-receptor for the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). Natural reactive antibodies (Abs) recognizing first loop (ECL1) of CCR5 have been detected in several pools of immunoglobulins from healthy donors and from several cohorts of either HIV-exposed but uninfected subjects (ESN) or HIV-infected individuals who control disease progression (LTNP) as well. The reason of development of anti-CCR5 Abs in the absence of autoimmune disease is still unknown; however, the presence of these Abs specific for CCR5 or for other immune receptors and mediators probably is related to homeostasis maintenance. The majority of anti-CCR5 Abs is directed to HIV binding site (N-terminus and ECL2) of the receptor. Conversely, it is well known that ECL1 of CCR5 does not bind HIV; thus, the anti-CCR5 Abs directed to ECL1 elicit a long-lasting internalization of CCR5 but not interfere with HIV binding directly; these Abs block HIV infection in either epithelial cells or CD4+ T lymphocytes and the mechanism differs from those ones described for all other CCR5-specific ligands. The Ab-mediated CCR5 internalization allows the formation of a stable signalosome by interaction of CCR5, ß-arrestin2 and ERK1 proteins. The signalosome degradation and the subsequent de novo proteins synthesis determine the CCR5 reappearance on the cell membrane with a very long-lasting kinetics (8 days). The use of monoclonal Abs to CCR5 with particular characteristics and mode of action may represent a novel mode to fight viral infection in either vaccinal or therapeutic strategies.

The ERK-1 function is required for HSV-1-mediated G1/S progression in HEP-2 cells and contributes to virus growth.

The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.

UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins.

Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.

Class B ß-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5.

CCR5 stimulation with natural ligands, such as RANTES, classically induces short-term internalization with transient activation of ß-arrestins and rapidly recycling on the cell surface. Here we discovered that, in T cells, natural CCR5 antibodies induce a CCR5-negative phenotype with the involvement of ß-arrestin2, which leads to the formation of a stable CCR5 signalosome with both ß-arrestin2 and ERK1. The activation of ß-arrestin2 is necessary to CCR5 signaling for the signalosome formation and stabilization. When all stimuli were washed out, ß-arrestin1 silencing favors the activity of ß-arrestin2 for the CCR5 signalosome retention. Interestingly, CCR5 turn from Class A trafficking pattern, normally used for its internalization with natural modulating molecules (i.e. RANTES), into a long lasting Class B type specifically induced by stimulation with natural anti-CCR5 antibodies. This new CCR5 pathway is relevant not only to study in depth the molecular basis of all pathologies where CCR5 is involved but also to generate new antidody-based therapeutics.

GM-3 Lactone Mimetic Interacts with CD4 and HIV-1 Env Proteins, Hampering HIV-1 Infection without Inducing a Histopathological Alteration.

Glycosphingolipids (GSLs) are involved in HIV-1 entry. GM-3 ganglioside, a widespread GSL, affects HIV entry and infection in different ways, depending on the concentration, through its anchoring activity in lipid rafts. This explains why the induction of an altered GSLs metabolism was a tempting approach to reducing HIV-1 cell infection. This study assayed the biological properties of a synthetic GM-3 lactone mimetic, 1, aimed at blocking HIV-1 infection without inducing the adverse events expected by an altered metabolism of GLSs in vivo. The mimetic, conjugated to immunogenic protein ovalbumin and multivalently presented, was able to bind the CD4 molecule with high affinity and block its engagement with gp120, thus inhibiting virus entry. Elicited antimimetic antibodies were also able to block HIV-1 infection in vitro, with activity complementary to that observed for 1. These preliminary results show that the use of GSLs mimetics can be a novel promising mode to block HIV-1 infection and that 1 and other GSL mimetics deserve further attention.

Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells.

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.