Biology subjects

Pizzato, M.

Publications and source records attributed to Pizzato, M..

4 recordsLinked to original sources

bioRxiv2025

SERINC5 Is the Primary Target of Retroviral Antagonists Among Human SERINC Paralogs and Requires a Tri-Cysteine Motif for Its Counteraction by HIV-1 Nef

The human transmembrane protein SERINC5 incorporates into budding HIV-1 particles and inhibits their fusion with new target cells. Among the five members of the SERINC family, SERINC3 and SERINC1 exhibit modest antiviral activity, whereas SERINC2 lacks this function. HIV-1 counteracts SERINC5 by promoting its endocytosis in a Nef- dependent manner. SERINC5 is also downregulated by glycoGag of Moloney murine leukaemia virus (MoMLV) and by the S2 glycoprotein of equine infectious anaemia virus (EIAV). Here, we demonstrate that all human SERINCs, despite their differing antiviral potencies, are incorporated into HIV-1 virions. SERINC5 is the most sensitive to viral antagonists, whereas its paralogs display moderate (SERINC2, SERINC3) or no (SERINC1) susceptibility, suggesting that SERINC5 has exerted stronger selective pressure on retroviral evolution. To identify the determinants of SERINC5 sensitivity to Nef, we performed structure- guided mutagenesis and identified a critical tri-cysteine motif (Cys355, Cys356, Cys358) within intracellular loop 4 (ICL4) required for Nef-mediated downregulation. Substitution of these residues with glutamines conferred resistance to Nef antagonism. While this motif is essential for SERINC5 internalization by diverse HIV-1 and SIV Nef alleles, it is dispensable for downregulation by MoMLV glycoGag and EIAV S2, indicating that these viral antagonists exploit distinct structural features of SERINC5. Finally, we show that SERINC5 is palmitoylated at the tri-cysteine motif, raising the possibility that this modification modulates its sensitivity to Nef. Together, these results confirm the central role of SERINC5 as a target of retroviral counteraction and highlight the crucial importance of ICL4 in mediating its engagement by Nef. ImportanceThe SERINC family of multipass transmembrane proteins includes host factors capable of restricting retrovirus infectivity. Among the five human paralogs, SERINC5 displays the strongest antiviral activity and is the only member efficiently targeted by all known retroviral antagonists, including HIV-1 Nef, MLV glycoGag, and the EIAV S2. This selectivity indicates that the evolutionary pressure exerted by SERINC proteins on retroviruses has been primarily driven by SERINC5 rather than by its relatives. Through structure-guided mutagenesis, we identified a tri-cysteine motif within intracellular loop 4 as a crucial determinant for SERINC5 downregulation by Nef, providing a molecular explanation for its distinctive susceptibility. These findings establish SERINC5 as the principal target of viral counteraction and suggest that its molecular features have contributed to shaping the adaptation of primate lentiviral Nef proteins to the host.

microbiology↗

bioRxiv2021

Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials. Author SummaryThe pandemic caused by the SARS-CoV-2 virus has created a completely unusual situation in rapidly searching for compounds able to interfere with the biological processes exploited by the virus. This new scenario has substantially changed the timing of drug development which has also resulted in the generation of controversial results, proving that the transition from computational screening to the clinical application requires great caution and careful studies. It is therefore necessary to establish new paradigms for evaluating the efficacy of a potential active molecule. We set up a preclinical platform aimed at identifying molecules active against SARS-CoV-2 infection developing a multidisciplinary approach based on very stringent experimental criteria, comprising in-silico studies, in vitro binding tests and infection studies with pseudovirus expressing the spike protein as well as clinically isolated SARS-CoV-2 strains. We focused our attention on doxycycline which has been suggested as potential therapeutic candidate for treating COVID-19 and is currently employed in about twenty clinical trials. Doxycycline resulted effective in inhibiting the transduction of pseudovirus but it did not affect the entry and replication of SARS-CoV-2. The results obtained underline the need to define more stringent and controlled pharmacological approaches before wasting precious time and resources with clinical trials.

pharmacology and toxicology↗

bioRxiv2021

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

immunology↗

bioRxiv2019

Influence of different glycoproteins and of the virion core on SERINC5 antiviral activity

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three accessory proteins encoded by diverse retroviruses, HIV-1 Nef, EIAV S2, and MLV Glycogag, each independently disrupt SERINC5 antiviral activity, by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., the vesicular stomatitis glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and M-PMV virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. Infectivity of particles, pseudotyped with HIV-1, amphotropic-MLV, or influenza virus glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. Particles generated by all three cores, and pseudotyped with glycoproteins from either avian leukosis virus-A, human endogenous retrovirus K (HERV-K), ecotropic-MLV, HTLV-1, Measles morbillivirus, lymphocytic choriomeningitis mammarenavirus (LCMV), Marburg virus, Ebola virus, severe acute respiratory syndrome-related coronavirus (SARS-CoV), or VSV, were insensitive to SERINC5. In contrast, particles pseudotyped with M-PMV, RD114, or rabies virus (RABV) glycoproteins were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 by particular glycoproteins did not correlate with reduced SERINC5 incorporation into particles or with the route of viral entry. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.\n\nIMPORTANCEThe importance of SERINC5 for inhibition of retroviruses is underscored by convergent evolution among three non-monophyletic retroviruses, each of which encodes a structurally unrelated SERINC5 inhibitor. One of these retroviruses causes tumors in mice, a second anemia in horses, and a third causes AIDS. SERINC5 is incorporated into retrovirus particles where it blocks entry into target cells, via a mechanism that is dependent on the viral glycoprotein. Here we demonstrate that retroviruses pseudotyped with glycoproteins from several non-retroviruses are also inhibited by SERINC5, suggesting that enveloped viruses other than retroviruses may also be inhibited by SERINC5. Additionally, we found that sensitivity to SERINC5 is determined by the retrovirus core, as well as by the glycoprotein. By better understanding how SERINC5 inhibits viruses we hope to extend fundamental understanding of virus replication and of the native role of SERINC5 in cells, and perhaps to advance the development of new antiviral strategies.

microbiology↗