Molecular Mechanisms Involved in the B Cell Growth and Clonogenic Activity of HIV-1 Matrix Protein p17 Variants.

The human immunodeficiency virus (HIV-1) matrix protein p17 (p17) is released from infected cells as a protein capable of deregulating the biological activity of different cells. P17 variants (vp17s), more frequently detected in the plasma of HIV-1+ patients with rather than without lymphoma and characterized by amino acids insertions in their C-terminal region, were found to trigger B cell growth and clonogenicity. Vp17s endowed with B-cell-growth-promoting activity are drastically destabilized, whereas, in a properly folded state, reference p17 (refp17) does not exert any biological activity on B cell growth and clonogenicity. However, misfolding of refp17 is necessary to expose a masked functional epitope, interacting with the protease-activated receptor 1 (PAR-1), endowed with B cell clonogenicity. Indeed, it is worth noting that changes in the secondary structure can strongly impact the function of a protein. Here, we performed computational studies to show that the gain of function of vp17s is linked to dramatic conformational changes due to structural modification in the secondary-structure elements and in the rearrangement of the hydrogen bond (H-bond) network. In particular, all clonogenic vp17s showed the disengagement of two critical residues, namely Trp16 and Tyr29, from their hydrophobic core. Biological data showed that the mutation of Trp16 and Tyr29 to Ala in the refp17 backbone, alone or in combination, resulted in a protein endowed with B cell clonogenic activity. These data show the pivotal role of the hydrophobic component in maintaining refp17 stability and identify a novel potential therapeutic target to counteract vp17-driven lymphomagenesis in HIV-1+ patients.

Epidemiology and molecular characterization of respiratory viruses at the end of COVID-19 pandemic in Lombardy, Northern Italy.

The COVID-19 pandemic forced the adoption of non-pharmaceutical interventions (NPIs) which influenced the circulation of other respiratory pathogens, such as Influenza virus (FLU), Parainfluenza virus (PIV), Respiratory Syncytial virus (RSV), Rhinovirus (RV), Enterovirus (EV), Adenovirus (AdV), Human Metapneumovirus (hMPV), and Human Coronavirus (CoV). The aim of the current study was to investigate how, with the end of the pandemic, the withdrawal of the NPIs impacted on the circulation and distribution of common respiratory viruses. The analyzed samples were collected from June 2021 to March 2023 (post-pandemic period) and compared to ones from the pandemic period. Nucleic acid detection of all respiratory viruses was performed by multiplex real time Polymerase Chain Reaction (PCR) and sequencing was conducted by Next Generation Sequencing (NGS) technique. Our analysis shows that the NPIs adopted against SARS-CoV-2 were also effective in controlling the spread of other respiratory viruses. Moreover, we documented how RV/EVs were the most commonly identified species, with the more abundant strains represented by Coxsackievirus (CV)-A/B and RV-A/C. RV/EVs were also detected in some co-infection cases; in particular, the majority of co-infections concerned CV-B/RV-A, CV-B/ECHO. Given the pandemic potential of respiratory viruses, accurate molecular screening is essential for a proper surveillance and prevention strategy.

Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

Nutritional Strategies To Improve VRE Control.

The rise of Vancomycin-resistant enterococci (VRE) strains among cellular therapy recipients raises concerns due to increased morbidity, mortality, and hospitalization costs, particularly impacting transplanted patients with diminished survival expectations. Recent research linking lactose to Enterococcus growth and graft-versus-host disease (GVHD) emphasizes the need for data on reducing lactose in the diets of VRE-carrying patients, especially in cellular therapy contexts like CAR-T or allogeneic hematopoietic stem cell transplantation. Responding to elevated VRE positivity rates in rectal swabs among patients in our BMT Unit, a unique nutritional strategy was implemented, introducing lactose-free milk and strictly enforcing lactose-free diets. This approach resulted in a significant reduction in VRE carriers, with a 16% positivity rate in the Lactose Group versus 3.6% in the Lactose-Free Group, as of June 2023. These results indicate the potential efficacy of this innovative nutritional strategy in high-risk departments, such as BMT Units and Intensive Care Units, with implications for reducing isolation strategies and inappropriate antibiotic use in cases of VRE colonization.

Detection of HIV-1 matrix protein p17 in sera of viremic and aviremic patients.

People living with human immunodeficiency virus type 1 (HIV-1), even if successfully treated with a combined antiretroviral therapy, display a persistent inflammation and chronic immune activation, and an increasing risk of developing cardiovascular and thrombotic events, cancers, and neurologic disorders. Accumulating evidence reveals that biologically active HIV-1 proteins may play a role in the development of these HIV-1-associated conditions. The HIV-1 matrix protein p17 (p17) is released and accumulates in different organs and tissue where it may exert multiple biological activities on different target cells. To assess a role of p17 in different HIV-1-related pathological processes, it is central to definitively ascertain and quantitate its expression in a large number of sera obtained from HIV-1-infected (HIV-1+) patients. To this aim, we developed a specific and highly sensitive p17 capture immunoenzymatic assay. Data obtained highlight a heterogeneous expression of p17 in blood of tested patients, with patients who were negative or displayed from low to relatively high p17 blood concentrations (range from 0.05 to 7.29 nM). Moreover, we found that blood p17 concentration was totally independent from the viremic status of the patient. This finding calls for monitoring HIV-1+ patients in order to evaluate a possible correlation between p17 amount in blood and the likelihood of developing HIV-1-related pathological conditions.

Plasma EBV DNA as a prognostic factor in EBV associated gastric cancer: a multicenter, prospective study (EBV PRESAGE study).

Purpose: The Cancer Genome Atlas Research Network identified Epstein-Barr-Virus (EBV)-positive gastric cancer as a distinct molecular subtype. The prevalence is 8-9% and the histological examination shows pronounced lymphocytic infiltration, elevated levels of IFN-γ and consequently overexpression of PD-L1. The role of plasma EBV DNA load as a prognostic factor in patients with this cancer subtype is still to be defined. Methods and analysis: The present multicenter prospective observational study "EBV PRESAGE", involving German and Italian cancer centers, aims to evaluate the prognostic role of plasma EBV DNA in EBV-related gastric cancer (GC). The objective is to study the association between plasma EBV DNA load at different consecutive time points and the patient's prognosis. Every patient with a new diagnosis of gastric cancer (including gastroesophageal junction adenocarcinoma) will be screened for Epstein-Barr encoded small Region (EBER) on tissue biopsies using in situ hybridization (ISH). If EBER ISH is positive, blood analysis for plasma EBV DNA will be conducted. The plasma EBV quantitative analysis will be centralized, and extraction, detection, and quantification of EBV DNA in plasma samples will be performed using real-time PCR. Discussion: We hypothesized that plasma EBV DNA represents a non-invasive tool for monitoring EBV-related GC and might be valuable as a prognostic marker.

DHFR Inhibitors Display a Pleiotropic Anti-Viral Activity against SARS-CoV-2: Insights into the Mechanisms of Action.

During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

First Italian report of a liver abscess and metastatic endogenous endophthalmitis caused by ST-23 hypervirulent Klebsiella pneumoniae in an immunocompetent individual.

BACKGROUND: Klebsiella pneumoniae is a common species in the gut of mammals and is widely distributed in the environment. However, the environmental source of hvKp that precedes gut colonization is unclear, but once that it reaches the gut there is a possible generalized spread y fecal-oral transmission especially in endemic areas. Liver abscess might develop when the bacteria, using its virulence factors, cross the intestinal barrier and invade the liver by the portal circulation. This syndrome, prevalent mostly in Asian countries, is increasingly reported in Western Countries and leaves open questions about the source of infection. CASE: Here we describe for the first time in Italy, a case of pyogenic liver abscess caused by a hypervirulent Klebsiella pneumoniae (HvKp) complicated by endophthalmitis and other metastatic infections in lung and prostate in an immunocompetent Chinese healthy individual with no recent travel in Asia. CONCLUSION: This case underlines the need for increased awareness of hypervirulent K. pneumoniae, even in settings where it occurs infrequently and where there are not evident epidemiological links.

Successful CAR-T cell therapy in a refractory MCL patient with bacterial, fungal and COVID-19 infection: a case report.

Background: The COVID-19 pandemic has had a significant impact on the management and care of onco-hematological patients, particularly those with lymphoproliferative disorders who are at higher risk for COVID-19 associated bacterial and fungal superinfections. Case presentation: We present the successful treatment of a 44-year-old male patient with refractory mantle cell lymphoma treated with chimeric antigen receptor T (CAR-T) cell therapy, despite concurrent COVID-19 infection. The patient developed grade II cytokine release syndrome, requiring admission to the intensive care unit. The CAR-T cells expanded effectively, and the patient achieved complete metabolic remission. During the treatment course, the patient experienced complications including COVID-19-associated pulmonary aspergillosis and a co-infection with Stenotrophomonas maltophilia and the SARS-CoV-2 omicron variant. Prompt antifungal and antibacterial therapy, along with appropriate COVID-19 treatment, led to the resolution of these infections. Dexamethasone was also administered to reduce inflammation and aid hematologic recovery. Despite the presence of multiple infections, the patient achieved complete remission of lymphoma, highlighting the effectiveness of CAR-T cell therapy in this high-risk patient. Conclusion: Despite the challenges posed by concurrent infections, the decision to proceed with CAR-T cell therapy in this patient proved to be successful, resulting in complete remission of lymphoma. Early initiation of supportive therapies and the use of dexamethasone contributed to the resolution of complications. This case underscores the importance of individualized decision-making and the potential benefits of CAR-T cell therapy in similar high-risk patients.

Results of an Innovative Program for Surveillance, Prophylaxis, and Treatment of Infectious Complications Following Allogeneic Stem Cell Transplantation in Hematological Malignancies (BATMO Protocol).

Background: Infectious complications are a significant cause of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplantation (Allo-SCT). The BATMO (Best-Antimicrobial-Therapy-TMO) is an innovative program for infection prevention and management and has been used in our centre since 2019. The specific features of the BATMO protocol regard both prophylaxis during neutropenia (abandonment of fluoroquinolone, posaconazole use in high-risk patients, aerosolized liposomal amphotericin B use until engraftment or a need for antifungal treatment, and letermovir use in CMV-positive recipients from day 0 to day +100) and therapy (empirical antibiotics based on patient clinical history and colonization, new antibiotics used in second-line according to antibiogram with the exception of carbapenemase-producing K pneumoniae for which the use in first-line therapy is chosen). Methods: Data on the infectious complications of 116 transplant patients before BATMO protocol (Cohort A; 2016 - 2018) were compared to those of 84 transplant patients following the introduction of the BATMO protocol (Cohort B; 2019 - 2021). The clinical and transplant characteristics of the 2 Cohorts were comparable, even though patients in Cohort B were at a higher risk of developing bacterial, fungal, and CMV infections, due to a significantly higher proportion of myeloablative regimens and haploidentical donors. Results: No change in the incidence of infections with organ localization was observed between the two Cohorts. A significant reduction in Clostridioides difficile infections by day +100 was observed in Cohort B (47% vs. 15%; p=0.04). At day +30, a higher incidence of Gram-negative bloodstream infections (BSIs) was observed in Cohort B (12% vs. 23%; p=0.05). By day +100 and between days +100 and +180, the incidence of BSIs and of the various etiological agents, the mortality from Gram-negative bacteria, and the incidence of invasive fungal infections were not different in the two Cohorts. The incidence of CMV reactivations by day +100 dropped drastically in patients of Cohort B, following letermovir registration (51% vs. 15%; p=0.00001). Discussion: The results of this study suggest that the BATMO program is safe. In particular, the choice to avoid prophylaxis with fluoroquinolone was associated with an increase in Gram-negative BSIs by day +30, but this did not translate into higher levels of mortality. Moreover, this strategy was associated with a significant reduction of Clostridiodes difficile infections. The efficacy of anti-CMV prophylaxis with letermovir was confirmed by a significant reduction in CMV reactivations. Even though patients in Cohort B were at higher risk of developing fungal infections (more haploidentical transplants with more myeloablative regimens), the extensive use of posaconazole for prophylaxis balanced this risk, and no increase in the incidence of fungal-associated complications was observed.

HIV-1 mutants expressing B cell clonogenic matrix protein p17 variants are increasing their prevalence worldwide.

AIDS-defining cancers declined after combined antiretroviral therapy (cART) introduction, but lymphomas are still elevated in HIV type 1 (HIV-1)-infected patients. In particular, non-Hodgkin's lymphomas (NHLs) represent the majority of all AIDS-defining cancers and are the most frequent cause of death in these patients. We have recently demonstrated that amino acid (aa) insertions at the HIV-1 matrix protein p17 COOH-terminal region cause protein destabilization, leading to conformational changes. Misfolded p17 variants (vp17s) strongly impact clonogenic B cell growth properties that may contribute to B cell lymphomagenesis as suggested by the significantly higher frequency of detection of vp17s with COOH-terminal aa insertions in plasma of HIV-1-infected patients with NHL. Here, we expand our previous observations by assessing the prevalence of vp17s in large retrospective cohorts of patients with and without lymphoma. We confirm the significantly higher prevalence of vp17s in lymphoma patients than in HIV-1-infected individuals without lymphoma. Analysis of 3,990 sequences deposited between 1985 and 2017 allowed us to highlight a worldwide increasing prevalence of HIV-1 mutants expressing vp17s over time. Since genomic surveillance uncovered a cluster of HIV-1 expressing a B cell clonogenic vp17 dated from 2011 to 2019, we conclude that aa insertions can be fixed in HIV-1 and that mutant viruses displaying B cell clonogenic vp17s are actively spreading.

Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions.

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms-in terms of cold and mechanical allodynia-and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.

Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

CD146+ Pericytes Subset Isolated from Human Micro-Fragmented Fat Tissue Display a Strong Interaction with Endothelial Cells: A Potential Cell Target for Therapeutic Angiogenesis.

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2+/PDGFRß+/CD105+ cells. Here, we tested the fat-derived PCs for the dispensability of the CD146 marker with the aim of better understanding the role of these PC subpopulations on angiogenesis. Cells from both CD146-positive (CD146+) and negative (CD146-) populations were observed to interact with human umbilical vein ECs (HUVECs). In addition, fat-derived PCs were able to induce angiogenesis of ECs in spheroids assay; and conditioned medium (CM) from both PCs and fat tissue itself led to the proliferation of ECs, thereby marking their role in angiogenesis stimulation. However, we found that CD146+ cells were more responsive to PDGF-BB-stimulated migration, adhesion, and angiogenic interaction with ECs, possibly owing to their higher expression of NCAM/CD56 than the corresponding CD146- subpopulation. We conclude that in fat tissue, CD146-expressing cells may represent a more mature pericyte subpopulation that may have higher efficacy in controlling and stimulating vascular regeneration and stabilization than their CD146-negative counterpart.

Management of Invasive Infections due to a Rare Arthroconidial Yeast, Saprochaete capitata, in Two Patients with Acute Hematological Malignancies.

Saprochaete capitata is an arthroconidial yeast, found principally in the environment, even if it belongs also to the normal microbial flora that colonize human subjects. This yeast is increasingly associated with invasive infections in hematological patients, in particular in those affected by acute leukemia. An important risk factor that predisposes to this infection is the profound neutropenia present in such immunocompromised patients. Saprochaete spp. were found resistant to both echinocandins and fluconazole so the treatment is often difficult. Here, we report two cases of sepsis in two patients with acute leukemia. All of them had fatal events, due to the worsening of their clinical condition. An early diagnosis and appropriate management of these pathogens is important in consideration of the poor prognosis associated to these fungal invasive infections.

Single-Shot Local Injection of Microfragmented Fat Tissue Loaded with Paclitaxel Induces Potent Growth Inhibition of Hepatocellular Carcinoma in Nude Mice.

Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers.

Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study.

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017-February 2020 (pre-pandemic period) and March 2020-May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

Binding to PI(4,5)P2 is indispensable for secretion of B-cell clonogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1-seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala-Ala) or 125 to 126 (Gly-Asn), respectively, are secreted from HIV-1-infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing.