PAD-mediated citrullination is a novel candidate diagnostic marker and druggable target for HPV-associated cervical cancer.

Citrullination is an emerging post-translational modification catalyzed by peptidyl-arginine deiminases (PADs) that convert peptidyl-arginine into peptidyl-citrulline. In humans, the PAD family consists of five isozymes (PADs 1-4, 6) involved in multiple diseases, including cancer. Given that high-risk (hr) human papillomaviruses (HPVs) are the etiological agents of cervical cancer, in this study, we sought to determine whether PAD-mediated protein citrullination would play a functional role in the HPV-driven transformation of epithelial cells. Here we show that both total protein citrullination and PAD4 expression levels are significantly associated with cervical cancer progression. Specifically, epithelial immunostaining for PAD4 revealed an increasingly higher histoscore from low-grade (CIN1) to high-grade (CIN2, CIN3) cervical intraepithelial neoplasia, and invasive squamous cell carcinoma (SCC) lesions, raising the attractive possibility that PAD4 may be used as tumor staging markers. Furthermore, taking advantage of the epidermoid cervical cancer cell line CaSki, which harbors multiple copies of the integrated HPV16 genome, we show that the expression of E6 and E7 HPV oncoproteins is impaired by treatment with the pharmacological pan-PAD inhibitor BB-Cl-amidine. Consistently, p53 and p21, two targets of HPV oncoproteins, are upregulated by the PAD inhibitor, which undergoes cell growth arrest and apoptosis. Altogether, these findings highlight a novel mechanism by which hrHPVs alter host regulatory pathways involved in cell cycle and survival to gain viral fitness, raising the possibility that PADs may represent an attractive target for developing novel host-targeting antivirals effective in preventing cervical cancer progression.

Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

Antiherpetic Activity of a Root Exudate from Solanum lycopersicum.

The rise of drug resistance to antivirals poses a significant global concern for public health; therefore, there is a pressing need to identify novel compounds that can effectively counteract strains resistant to current antiviral treatments. In light of this, researchers have been exploring new approaches, including the investigation of natural compounds as alternative sources for developing potent antiviral therapies. Thus, this work aimed to evaluate the antiviral properties of the organic-soluble fraction of a root exudate derived from the tomato plant Solanum lycopersicum in the context of herpesvirus infections. Our findings demonstrated that a root exudate from Solanum lycopersicum exhibits remarkable efficacy against prominent members of the family Herpesviridae, specifically herpes simplex virus type 1 (HSV-1) (EC50 25.57 µg/mL, SI > 15.64) and human cytomegalovirus (HCMV) (EC50 9.17 µg/mL, SI 32.28) by inhibiting a molecular event during the herpesvirus replication phase. Moreover, the phytochemical fingerprint of the Solanum lycopersicum root exudate was characterized through mass spectrometry. Overall, these data have unveiled a novel natural product with antiherpetic activity, presenting a promising and valuable alternative to existing drugs.

Citrullination profile analysis reveals peptidylarginine deaminase 3 as an HSV-1 target to dampen the activity of candidate antiviral restriction factors.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual's life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer's disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

Strigolactones as Broad-Spectrum Antivirals against ß-Coronaviruses through Targeting the Main Protease Mpro.

The current SARS-CoV-2 pandemic and the likelihood that new coronavirus strains will emerge in the immediate future point out the urgent need to identify new pan-coronavirus inhibitors. Strigolactones (SLs) are a class of plant hormones with multifaceted activities whose roles in plant-related fields have been extensively explored. Recently, we proved that SLs also exert antiviral activity toward herpesviruses, such as human cytomegalovirus (HCMV). Here we show that the synthetic SLs TH-EGO and EDOT-EGO impair ß-coronavirus replication including SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Interestingly, in silico simulations suggest the binding of SLs in the SARS-CoV-2 main protease (Mpro) active site, and this was further confirmed by an in vitro activity assay. Overall, our results highlight the potential efficacy of SLs as broad-spectrum antivirals against ß-coronaviruses, which may provide the rationale for repurposing this class of hormones for the treatment of COVID-19 patients.

IFI16 Impacts Metabolic Reprogramming during Human Cytomegalovirus Infection.

Cellular lipid metabolism plays a pivotal role in human cytomegalovirus (HCMV) infection, as increased lipogenesis in HCMV-infected cells favors the envelopment of newly synthesized viral particles. As all cells are equipped with restriction factors (RFs) able to exert a protective effect against invading pathogens, we asked whether a similar defense mechanism would also be in place to preserve the metabolic compartment from HCMV infection. Here, we show that gamma interferon (IFN-γ)-inducible protein 16 (IFI16), an RF able to block HCMV DNA synthesis, can also counteract HCMV-mediated metabolic reprogramming in infected primary human foreskin fibroblasts (HFFs), thereby limiting virion infectivity. Specifically, we find that IFI16 downregulates the transcriptional activation of the glucose transporter 4 (GLUT4) through cooperation with the carbohydrate-response element-binding protein (ChREBP), thereby reducing HCMV-induced transcription of lipogenic enzymes. The resulting decrease in glucose uptake and consumption leads to diminished lipid synthesis, which ultimately curbs the de novo formation of enveloped viral particles in infected HFFs. Consistently, untargeted lipidomic analysis shows enhanced cholesteryl ester levels in IFI16 KO versus wild-type (WT) HFFs. Overall, our data unveil a new role of IFI16 in the regulation of glucose and lipid metabolism upon HCMV replication and uncover new potential targets for the development of novel antiviral therapies. IMPORTANCE Human cytomegalovirus (HCMV) gathers all the substrates and enzymes necessary for the assembly of new virions from its host cell. For instance, HCMV is known to induce cellular metabolism of infected cells to favor virion assembly. Cells are, however, equipped with a first-line defense represented by restriction factors (RFs), which after sensing viral DNA can trigger innate and adaptive responses, thereby blocking HCMV replication. One such RF is IFN-γ-inducible protein 16 (IFI16), which we have shown to downregulate viral replication in human fibroblasts. Thus, we asked whether IFI16 would also play a role in preserving cellular metabolism upon HCMV infection. Our findings highlight an unprecedented role of IFI16 in opposing the metabolic changes elicited by HCMV, thus revealing new promising targets for antiviral therapy.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2.

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Human cytomegalovirus-induced host protein citrullination is crucial for viral replication.

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.

Human Cytomegalovirus and Autoimmune Diseases: Where Are We?

Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus belonging to the ß-subgroup of the herpesvirus family. After the initial infection, the virus establishes latency in poorly differentiated myeloid precursors from where it can reactivate at later times to cause recurrences. In immunocompetent subjects, primary HCMV infection is usually asymptomatic, while in immunocompromised patients, HCMV infection can lead to severe, life-threatening diseases, whose clinical severity parallels the degree of immunosuppression. The existence of a strict interplay between HCMV and the immune system has led many to hypothesize that HCMV could also be involved in autoimmune diseases (ADs). Indeed, signs of active viral infection were later found in a variety of different ADs, such as rheumatological, neurological, enteric disorders, and metabolic diseases. In addition, HCMV infection has been frequently linked to increased production of autoantibodies, which play a driving role in AD progression, as observed in systemic lupus erythematosus (SLE) patients. Documented mechanisms of HCMV-associated autoimmunity include molecular mimicry, inflammation, and nonspecific B-cell activation. In this review, we summarize the available literature on the various ADs arising from or exacerbating upon HCMV infection, focusing on the potential role of HCMV-mediated immune activation at disease onset.

HPV Meets APOBEC: New Players in Head and Neck Cancer.

Besides smoking and alcohol, human papillomavirus (HPV) is a factor promoting head and neck squamous cell carcinoma (HNSCC). In some human tumors, including HNSCC, a number of mutations are caused by aberrantly activated DNA-modifying enzymes, such as the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases. As the enzymatic activity of APOBEC proteins contributes to the innate immune response to viruses, including HPV, the role of APOBEC proteins in HPV-driven head and neck carcinogenesis has recently gained increasing attention. Ongoing research efforts take the cue from two key observations: (1) APOBEC expression depends on HPV infection status in HNSCC; and (2) APOBEC activity plays a major role in HPV-positive HNSCC mutagenesis. This review focuses on recent advances on the role of APOBEC proteins in HPV-positive vs. HPV-negative HNSCC.

Where do we Stand after Decades of Studying Human Cytomegalovirus?

Human cytomegalovirus (HCMV), a linear double-stranded DNA betaherpesvirus belonging to the family of Herpesviridae, is characterized by widespread seroprevalence, ranging between 56% and 94%, strictly dependent on the socioeconomic background of the country being considered. Typically, HCMV causes asymptomatic infection in the immunocompetent population, while in immunocompromised individuals or when transmitted vertically from the mother to the fetus it leads to systemic disease with severe complications and high mortality rate. Following primary infection, HCMV establishes a state of latency primarily in myeloid cells, from which it can be reactivated by various inflammatory stimuli. Several studies have shown that HCMV, despite being a DNA virus, is highly prone to genetic variability that strongly influences its replication and dissemination rates as well as cellular tropism. In this scenario, the few currently available drugs for the treatment of HCMV infections are characterized by high toxicity, poor oral bioavailability, and emerging resistance. Here, we review past and current literature that has greatly advanced our understanding of the biology and genetics of HCMV, stressing the urgent need for innovative and safe anti-HCMV therapies and effective vaccines to treat and prevent HCMV infections, particularly in vulnerable populations.

Tuning the Orchestra: HCMV vs. Innate Immunity.

Understanding how the innate immune system keeps human cytomegalovirus (HCMV) in check has recently become a critical issue in light of the global clinical burden of HCMV infection in newborns and immunodeficient patients. Innate immunity constitutes the first line of host defense against HCMV as it involves a complex array of cooperating effectors - e.g., inflammatory cytokines, type I interferon (IFN-I), natural killer (NK) cells, professional antigen-presenting cells (APCs) and phagocytes - all capable of disrupting HCMV replication. These factors are known to trigger a highly efficient adaptive immune response, where cellular restriction factors (RFs) play a major gatekeeping role. Unlike other innate immunity components, RFs are constitutively expressed in many cell types, ready to act before pathogen exposure. Nonetheless, the existence of a positive regulatory feedback loop between RFs and IFNs is clear evidence of an intimate cooperation between intrinsic and innate immunity. In the course of virus-host coevolution, HCMV has, however, learned how to manipulate the functions of multiple cellular players of the host innate immune response to achieve latency and persistence. Thus, HCMV acts like an orchestra conductor able to piece together and rearrange parts of a musical score (i.e., innate immunity) to obtain the best live performance (i.e., viral fitness). It is therefore unquestionable that innovative therapeutic solutions able to prevent HCMV immune evasion in congenitally infected infants and immunocompromised individuals are urgently needed. Here, we provide an up-to-date review of the mechanisms regulating the interplay between HCMV and innate immunity, focusing on the various strategies of immune escape evolved by this virus to gain a fitness advantage.

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.