HIV and FIV glycoproteins increase cellular tau pathology via cGMP-dependent kinase II activation

As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically improves the lifespan of individuals with HIV, many are now entering the prime age when Alzheimer's disease (AD)-like symptoms begin to manifest. Hyperphosphorylated tau, a known AD pathological characteristic, has been prematurely increased in the brains of HIV-infected patients as early as in their 30s and is increased with age. This thus suggests that HIV infection may lead to accelerated AD phenotypes. However, whether HIV infection causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have previously revealed that viral glycoproteins, HIV gp120 and feline immunodeficiency virus (FIV) gp95, induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons to demonstrate that HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.

Fusexins, HAP2/GCS1 and Evolution of Gamete Fusion

Gamete fusion is the climax of fertilization in all sexually reproductive organisms, from unicellular fungi to humans. Similarly to other cell-cell fusion events, gamete fusion is mediated by specialized proteins, named fusogens, that overcome the energetic barriers during this process. In recent years, HAPLESS 2/GENERATIVE CELL-SPECIFIC 1 (HAP2/GCS1) was identified as the fusogen mediating sperm-egg fusion in flowering plants and protists, being both essential and sufficient for the membrane merger in some species. The identification of HAP2/GCS1 in invertebrates, opens the possibility that a similar fusogen may be used in vertebrate fertilization. HAP2/GCS1 proteins share a similar structure with two distinct families of exoplasmic fusogens: the somatic Fusion Family (FF) proteins discovered in nematodes, and class II viral glycoproteins (e.g., rubella and dengue viruses). Altogether, these fusogens form the Fusexin superfamily. While some attributes are shared among fusexins, for example the overall structure and the possibility of assembly into trimers, some other characteristics seem to be specific, such as the presence or not of hydrophobic loops or helices at the distal tip of the protein. Intriguingly, HAP2/GCS1 or other fusexins have neither been identified in vertebrates nor in fungi, raising the question of whether these genes were lost during evolution and were replaced by other fusion machinery or a significant divergence makes their identification difficult. Here, we discuss the biology of HAP2/GCS1, its involvement in gamete fusion and the structural, mechanistic and evolutionary relationships with other fusexins.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.

Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

Mutagenic Distinction between the Receptor-Binding and Fusion Subunits of the SARS-CoV-2 Spike Glycoprotein and Its Upshot

We observe that a residue R of the spike glycoprotein of SARS-CoV-2 that has mutated in one or more of the current variants of concern or interest, or under monitoring, rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A related study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron.

Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein

We observe that a residue R of the spike glycoprotein of SARS-CoV-2 which has mutated in one or more of the current Variants of Concern or Interest and under Monitoring rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A possible explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally.

Using the vaccinia virus MVA strain for developing recombinant vector vaccines against current arboviral infections

Epidemic vector-borne viral infections pose a serious threat to public health worldwide. There is currently no specific preventive treatment for most of them. One of the promising solutions for combating viral fevers is development of vector vaccines, including MVA-based vaccines, which have virtually no adverse side effects. The safety of the MVA strain and absent reactogenicity of recombinant MVA vaccines have been supported by many clinical trials.The article focuses on test results for similar preventive products against viral fevers: Crimean-Congo hemorrhagic fever, Rift Valley fever, yellow fever, Chikungunya and Zika fevers.Their immunogenicity was evaluated on immunocompetent and immunocompromised white mice; their protective efficacy was assessed on immunocompromised white mice deficient in IFN-α/β receptors, that are used for experimental modeling of the infection. Nearly all the new recombinant vaccines expressing immunodominant antigens demonstrated 100% protective efficacy. It has been found that although the vaccine expressing Zika virus structural proteins induced antibodies against specific viral glycoproteins, it can be associated with high risks when used for prevention of Zika fever in individuals who had dengue fever in the past, due to the phenomenon known as antibody-dependent enhancement of infection, which can occur in diseases caused by antigenically related flaviruses. For this reason, the vaccine expressing non-structural protein 1 (NS1) was developed for vaccination against Zika fever.The yellow fever vaccine developed on the MVA platform had immunogenicity similar to that of the commercial 17D vaccine, outperforming the latter in safety.

Characterization of Natural and Synthetic Sialoglycans Targeting the Hemagglutinin-Neuraminidase of Mumps Virus

The inhibition of surface viral glycoproteins offers great potential to hamper the attachment of viruses to the host cells surface and the spreading of viral infection. Mumps virus (MuV) is the etiological agent of the mumps infectious disease and causes a wide spectrum of mild to severe symptoms due to the inflammation of the salivary glands. Here we focus our attention on the hemagglutinin-neuraminidase (HN) isolated from MuV SBL-1 strain. We describe the molecular features of host sialoglycans recognition by HN protein by means of NMR, fluorescence assays and computational studies. Furthermore, we also describe the synthesis of a N-acetylneuraminic acid-derived thiotrisaccharide targeting the viral protein, and the corresponding 3D-complex. Our results provide the basis to improve the design and synthesis of potent viral hemagglutinin-neuraminidase inhibitors.

Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine

The COVID-19 pandemic has brought to the forefront an urgent need for the rapid development of highly efficacious vaccines, particularly in light of the ongoing emergence of multiple variants of concern. Plant-based recombinant protein platforms are emerging as cost-effective and highly scalable alternatives to conventional protein production. Viral glycoproteins, however, are historically challenging to produce in plants. Herein, we report the production of plant-expressed wild-type glycosylated SARS-CoV-2 Spike RBD (receptor-binding domain) protein that is recognized by anti-RBD antibodies and exhibits high-affinity binding to the SARS-CoV-2 receptor ACE2 (angiotensin-converting enzyme 2). Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from naturally infected convalescent, vaccinated, or convalescent and vaccinated individuals. We further demonstrate that RBD binding to the ACE2 receptor was efficiently neutralized by antibodies from sera of SARS-CoV-2 convalescent and partially and fully vaccinated individuals. Collectively, these findings demonstrate that recombinant RBD produced in planta exhibits suitable biochemical and antigenic features for use in a subunit vaccine platform.

Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1β. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1β release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.