Heparin Precursors with Reduced Anticoagulant Properties Retain Antiviral and Protective Effects That Potentiate the Efficacy of Sofosbuvir against Zika Virus Infection in Human Neural Progenitor Cells.

Zika virus (ZIKV) infection during pregnancy can result in severe birth defects, such as microcephaly, as well as a range of other related health complications. Heparin, a clinical-grade anticoagulant, is shown to protect neural progenitor cells from death following ZIKV infection. Although heparin can be safely used during pregnancy, it retains off-target anticoagulant effects if directly employed against ZIKV infection. In this study, we investigated the effects of chemically modified heparin derivatives with reduced anticoagulant activities. These derivatives were used as experimental probes to explore the structure-activity relationships. Precursor fractions of porcine heparin, obtained during the manufacture of conventional pharmaceutical heparin with decreased anticoagulant activities, were also explored. Interestingly, these modified heparin derivatives and precursor fractions not only prevented cell death but also inhibited the ZIKV replication of infected neural progenitor cells grown as neurospheres. These effects were observed regardless of the specific sulfation position or overall charge. Furthermore, the combination of heparin with Sofosbuvir, an antiviral licensed for the treatment of hepatitis C (HCV) that also belongs to the same Flaviviridae family as ZIKV, showed a synergistic effect. This suggested that a combination therapy approach involving heparin precursors and Sofosbuvir could be a potential strategy for the prevention or treatment of ZIKV infections.

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Origin and evolution of SARS-CoV-2.

SARS-CoV-2 is a novel coronavirus that emerged in China at the end of 2019 causing the severe disease known as coronavirus disease 2019 (COVID-19). SARS-CoV-2, as to the previously highly pathogenic human coronaviruses named SARS-CoV, the etiological agent of severe acute respiratory syndrome (SARS), has a zoonotic origin, although SARS-CoV-2 precise chain of animal-to-human transmission remains undefined. Unlike the 2002-2003 pandemic caused by SARS-CoV whose extinction from the human population was achieved in eight months, SARS-CoV-2 has been spreading globally in an immunologically naïve population in an unprecedented manner. The efficient infection and replication of SARS-CoV-2 has resulted in the emergence of viral variants that have become predominant posing concerns about their containment as they are more infectious with variable pathogenicity in respect to the original virus. Although vaccine availability is limiting severe disease and death caused by SARS-CoV-2 infection, its extinction is far to be close and predictable. In this regard, the emersion of the Omicron viral variant in November 2021 was characterized by humoral immune escape and it has reinforced the importance of the global monitoring of SARS-CoV-2 evolution. Given the importance of the SARS-CoV-2 zoonotic origin, it will also be crucial to monitor the animal-human interface to be better prepared to cope with future infections of pandemic potential.

Modulation of NBAS-Related Functions in the Early Response to SARS-CoV-2 Infection.

Upon infection, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is predicted to interact with diverse cellular functions, such as the nonsense-mediated decay (NMD) pathway, as suggested by the identification of the core NMD factor upframeshift-1 (UPF1) in the SARS-CoV-2 interactome, and the retrograde transport from the Golgi to the endoplasmic reticulum (ER) through the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where coronavirus assembly occurs. Here, we investigated the expression and localization of the neuroblastoma-amplified sequence (NBAS) protein, a UPF1 partner for the NMD at the ER, participating also in retrograde transport, and of its functional partners, at early time points after SARS-CoV-2 infection of the human lung epithelial cell line Calu3. We found a significant decrease of DExH-Box Helicase 34 (DHX34), suppressor with morphogenetic effect on genitalia 5 (SMG5), and SMG7 expression at 6 h post-infection, followed by a significant increase of these genes and also UPF1 and UPF2 at 9 h post-infection. Conversely, NBAS and other genes coding for NMD factors were not modulated. Known NMD substrates related to cell stress (Growth Arrest Specific 5, GAS5; transducin beta-like 2, TBL2; and DNA damage-inducible transcript 3, DDIT3) were increased in infected cells, possibly as a result of alterations in the NMD pathway and of a direct effect of the infection. We also found that the expression of unconventional SNARE in the ER 1, USE1 (p31) and Zeste White 10 homolog, ZW10, partners of NBAS in the retrograde transport function, significantly increased over time in infected cells. Co-localization of NBAS and UPF1 proteins did not change within 24 h of infection nor did it differ in infected versus non-infected cells at 1 and 24 h after infection; similarly, the co-localization of NBAS and p31 proteins was not altered by infection in this short time frame. Finally, both NBAS and UPF1 were found to co-localize with SARS-CoV-2 S and N proteins. Overall, these data are preliminary evidence of an interaction between NBAS and NBAS-related functions and SARS-CoV-2 in infected cells, deserving further investigation.

Heparin Protects Human Neural Progenitor Cells from Zika Virus-Induced Cell Death While Preserving Their Differentiation into Mature Neuroglial Cells.

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.

The Long and Winding Road Towards an HIV Cure.

In the summer of 1981, a new deadly disease suddenly emerged targeting young men having sexwith men (MSM); three years later, a new virus, an exogenous human retrovirus, later named humanimmunodeficiency virus (HIV), was demonstrated to be the causative agent of the new disease, theAcquired Immuno-Deficiency Syndrome (AIDS), affecting, in addition to MSM, also intravenousdrug users, hemophiliacs, heterosexual individuals and children born to infected mothers. AIDSremained a dead sentence for >95% infected individuals until 1996 when the first combinationantiretroviral therapy (cART) was shown to be effective saving the lives of countless people. Sincethen, cART has become extremely powerful and simpler to adhere (now down to one or two pillsa day). However, virus eradication ("Cure") has been achieved thus far only in two individuals asa result of stem cell transplantation by an immunologically compatible donor homozygote for theCCR5Δ32 mutation; CCR5 is indeed the major entry coreceptor for the virus together with theprimary receptor CD4. This represents the exception to the rule that none of the many experimentalattempts of eliminating or silencing the virus reservoir unaffected by cART has achieved a significantproof of concept. In this article we will describe the essential aspects of the viral reservoirs and thecurrent strategies to tackle it.

Host Restriction Factors Modulating HIV Latency and Replication in Macrophages.

In addition to CD4+ T lymphocytes, myeloid cells and, particularly, differentiated macrophages are targets of human immunodeficiency virus type-1 (HIV-1) infection via the interaction of gp120Env with CD4 and CCR5 or CXCR4. Both T cells and macrophages support virus replication, although with substantial differences. In contrast to activated CD4+ T lymphocytes, HIV-1 replication in macrophages occurs in nondividing cells and it is characterized by the virtual absence of cytopathicity both in vitro and in vivo. These general features should be considered in evaluating the role of cell-associated restriction factors aiming at preventing or curtailing virus replication in macrophages and T cells, particularly in the context of designing strategies to tackle the viral reservoir in infected individuals receiving combination antiretroviral therapy. In this regard, we will here also discuss a model of reversible HIV-1 latency in primary human macrophages and the role of host factors determining the restriction or reactivation of virus replication in these cells.

A Community Study of SARS-CoV-2 Detection by RT-PCR in Saliva: A Reliable and Effective Method.

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.

Jurkat-Derived (J-Lat, J1.1, and Jurkat E4) and CEM-Derived T Cell Lines (8E5 and ACH-2) as Models of Reversible Proviral Latency.

The introduction of combination antiretroviral therapy (cART) has switched HIV-1 infection from a lethal disease to a chronic one. Indeed, cART is a lifelong treatment since its interruption is always followed by a rapid rebound of viremia from both cellular and anatomical viral reservoirs where the integrated HIV-1 provirus remains transcriptionally silent or maintains low-levels of viral replication, thereby preventing HIV-1 eradication. As therapeutic approach, the "shock and kill" strategy has emerged with the main objective to reactivate HIV-1 transcription from latency by using latency reversing agents (LRAs) prior to kill the reactivated infected cells by improving host immune responses. In this context, the development of tools such as HIV-1 latently infected cell lines have drastically increased our knowledge about HIV-1 latency and how to counteract this highly heterogeneous phenomenon. In this chapter, we will describe several chronically HIV-1 infected T-lymphocytic cell lines as useful surrogate models to study reversible HIV-1 proviral latency in CD4+ T cells in vitro before approaching more complex and expensive models.

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible Proviral Latency.

As already discussed for T cell lines, also myeloid cell lines as served as the earliest models of chronic HIV infection. They were particularly relevant in the late 1980s and early 1990s when most experimental in vitro infections were based on laboratory-adapted "T-cell tropic" strains of HIV-1, such as LAI/IIIB or others, that later were found to rely upon CXCR4 as coreceptor for viral entry in addition to CD4 as primary receptor. Although primary macrophages do express CXCR4 together with CD4, virus replication is much less efficient than that observed with CCR5-using "macrophage-tropic" strains, as discussed separately in this book. Although different myeloid cell lines have been used to generate models of chronic HIV-1 infection that could be used to investigate features of proviral reactivation, as reviewed in (Cassol et al. J Leukoc Biol 80:1018-1030, 2006), two cell lines in particular have been broadly used and will be here discussed: the U937-derived U1 and HL-60-derived OM-10.1.

Human Monocyte-Derived Macrophages (MDM): Model 2.

In addition to CD4+ T cells, tissue-resident macrophages are target of productive HIV-1 infection. Unlike CD4+ T lymphocytes they are characterized by a substantial resistance to the cytopathic effects triggered by viral infection. This feature, in addition to their homeostatic self-renewal capacity, strongly support the hypothesis that macrophages could serve as an additional reservoir of persistently infected cells in individuals receiving combination antiretroviral therapy (cART).In order to study the peculiar aspects of HIV-1 infection of macrophages, human primary monocyte-derived macrophages (MDM) represent the most exploited model given the difficulty to obtain and maintain in culture for significant periods of time macrophages from different organs and tissues. Here we present a model of MDM differentiation achieved in the absence of addition of exogenous cytokines (such as GM-CSF, discussed in the previous chapter), that could be further investigated in term of cell polarization toward classic, proinflammatory "M1", or alternatively activated "M2" cells before or after infection. We will also discuss how to reinforce the M1-polarization protocol to obtain a reliable model of reversible latency of infectious HIV-1 in primary  M1-MDM.

Biobanking for COVID-19 research.

BACKGROUND: Biobanks are imperative infrastructures, particularly during outbreaks, when there is an obligation to acquire and share knowledge as quick as possible to allow for implementation of science-based preventive, diagnostic, prognostic, and therapeutic strategies. METHODS: We established a COVID-19 biobank with the aim of collecting high-quality and well-annotated human biospecimens, in the effort to understand the pathogenic mechanisms underlying COVID-19 and identify therapeutic targets (COVID-BioB, NCT04318366). Here we describe our experience and briefly review the characteristics of the biobanks for COVID-19 that have been so far established. RESULTS: A total of 46,677 samples have been collected from 913 participants (63.3% males, median [IQR] age 62.2 [51.2-74.0] years) since the beginning of the program. Most patients (66.9%) had been admitted to hospital for COVID-19, with a median length of stay of 15.0 (9.0-27.0) days. A minority of patients (13.3% of the total) had been admitted for other reasons and subsequently tested positive for SARS-CoV-2. The remainder were managed at home after being seen at the Emergency Department. CONCLUSIONS: Having a solid research infrastructure already in place, along with flexibility and adaptability to new requirements, allowed for the quick building of a COVID-19 biobank that will help expand and share the knowledge of SARS-CoV-2.

TRIM22. A Multitasking Antiviral Factor.

Viral invasion of target cells triggers an immediate intracellular host defense system aimed at preventing further propagation of the virus. Viral genomes or early products of viral replication are sensed by a number of pattern recognition receptors, leading to the synthesis and production of type I interferons (IFNs) that, in turn, activate a cascade of IFN-stimulated genes (ISGs) with antiviral functions. Among these, several members of the tripartite motif (TRIM) family are antiviral executors. This article will focus, in particular, on TRIM22 as an example of a multitarget antiviral member of the TRIM family. The antiviral activities of TRIM22 against different DNA and RNA viruses, particularly human immunodeficiency virus type 1 (HIV-1) and influenza A virus (IAV), will be discussed. TRIM22 restriction of virus replication can involve either direct interaction of TRIM22 E3 ubiquitin ligase activity with viral proteins, or indirect protein-protein interactions resulting in control of viral gene transcription, but also epigenetic effects exerted at the chromatin level.

HLA-E-restricted CD8+ T Lymphocytes Efficiently Control Mycobacterium tuberculosis and HIV-1 Coinfection.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8+ T cells in patients with Mycobacterium tuberculosis and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8+ T cells but not by HLA-E-restricted CD8+ T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8+ T cells could play a protective role in Mycobacterium tuberculosis and HIV-1 coinfection. HLA-E- and HLA-A2-restricted Mycobacterium tuberculosis-specific CD8+ T cells were tested in vitro for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and Mycobacterium tuberculosis coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8+ T cells and failure to restrict the growth of intracellular Mycobacterium tuberculosis. Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells were expanded in the circulation of patients with Mycobacterium tuberculosis/HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued in vitro by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells in patients with Mycobacterium tuberculosis/HIV-1 coinfection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.

Interferon-inducible TRIM22 contributes to maintenance of HIV-1 proviral latency in T cell lines.

The human immunodeficiency virus type-1 (HIV-1) establishes a state of latent infection in a small number of CD4+ T lymphocytes that, nonetheless, represent a major obstacle to viral eradication. We here show that Tripartite Motif-containing protein 22 (TRIM22), an epigenetic inhibitor of Specificity protein 1 (Sp1)-dependent HIV-1 transcription, is a relevant factor in maintaining a state of repressed HIV-1 expression at least in CD4+ T cell lines. By knocking-down (KD) TRIM22 expression, we observed an accelerated reactivation of a doxycycline (Dox)-controlled HIV-1 replication in the T lymphocytic SupT1 cell line. Furthermore, we here report for the first time that TRIM22 is a crucial factor for maintaining a state of HIV-1 quiescence in chronically infected ACH2 -T cell line while its KD potentiated HIV-1 expression in both ACH-2 and J-Lat 10.6 cell lines upon cell stimulation with either tumor necrosis factor-α (TNF-α) or histone deacetylase inhibitors (HDACi). In conclusion, TRIM22 is a novel determinant of HIV-1 latency, at least in T cell lines, thus representing a potential pharmacological target for strategies aiming at curtailing or silencing the pool of latently infected CD4+ T lymphocytes constituting the HIV-1 reservoir in individuals receiving combination antiretroviral therapy.

The ATP/P2X7 axis in human immunodeficiency virus infection of macrophages.

HIV-1 infects CD4+ T lymphocytes with a 'helper' function and myeloid cells, mostly tissue-resident macrophages. While infection of CD4 T lymphocytes in the absence of combination antiretroviral therapy (cART) leads to their depletion and to a profound immunodeficiency, macrophages are resistant to virus-induced cytopathicity and are a source of infectious virus, particularly in the central nervous system (CNS). Infected macrophages are characterized by accumulating newly formed viral particles (virions) in subcellular vacuoles defined as 'virus-containing compartments (VCC)', derived from invaginations of the plasma membrane, that are poorly accessible to antiretroviral agents and anti-HIV antibodies. Several factors favor the accumulation of HIV-1 virions in VCC in vitro, whereas extracellular ATP, via binding to its receptor P2X7, is the only agent described thus far as capable of triggering the rapid release of VCC-sequestered virions without simultaneously causing the death of infected macrophages. Thus, the eATP/P2X7 axis could be exploited to achieve a pharmacological control of VCC-associated viral reservoir in individuals under effective cART.

Reversible Human Immunodeficiency Virus Type-1 Latency in Primary Human Monocyte-Derived Macrophages Induced by Sustained M1 Polarization.

We have reported that short-term stimulation of primary human monocyte-derived macrophages (MDM) with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), i.e. M1 polarization, leads to a significant containment of virus replication. Here we show that M1-MDM restimulation with these cytokines 7 days after infection (M12 MDM) promoted an increased restriction of HIV-1 replication characterized by very low levels of virus production near to undetectable levels. In comparison to control and M1-MDM that were not restimulated, M12 MDM showed a stronger reduction of both total and integrated HIV DNA as well as of viral mRNA expression. M12 MDM were characterized by an upregulated expression of restriction factors acting at the level of reverse transcription (RT), including apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and APOBEC3G, but not SAM domain and HD domain-containing protein 1 (SAMHD1). M12 MDM also showed an increased expression of Class II Transactivator (CIITA) and Tripartite Motif22 (TRIM22), two negative regulators of proviral transcription, whereas expression and phosphorylation of transcriptional inducers of HIV-1, such as nuclear factor kB (NF-kB) and signal transducer and activator of transcription 1 (STAT1), were not impaired in these cells. The almost quiescent state of the infection in M12 MDM was promptly reversed by coculture with mitogen-stimulated leukocytes or cell incubation with their filtered culture supernatant. M12 MDM harbored replication-competent HIV-1 as virus spreading following cell stimulation was fully prevented by the RT inhibitor lamivudine/3TC. Selective reactivation of proviral expression in M12 MDM, but not in control or in M1-MDM that were not restimulated, was confirmed in cells infected with single round Vesicular Stomatitis Virus-G-pseudotyped HIV-1. Thus, M12 MDM represent an in vitro model of reversible, almost quiescent HIV-1 infection of primary human macrophages that could be further exploited for "Cure" related investigations.