Fusion of a New World Alphavirus with Membrane Microdomains Involving Partially Reversible Conformational Changes in the Viral Spike Proteins.

Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry mechanism. In this study, we show for the first time the interaction of an enveloped virus with membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon acidification, although its kinetics was quite slow when compared to that of fusion with artificial liposomes demonstrated in a previous work. Surprisingly, when virus was previously exposed to acidic pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well as infectivity, and then reneutralized, its ability to fuse with membrane microdomains at low pH was retained. Interestingly, this observation correlated with a partial reversion of low pH-induced conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our results suggest that MAYV entry into host cells could alternatively involve internalization via lipid rafts and that the conformational changes triggered by low pH in the viral spike proteins during the entry process are partially reversible.

Pressure-Inactivated Virus: A Promising Alternative for Vaccine Production.

In recent years, many applications in diverse scientific fields with various purposes have examined pressure as a thermodynamic parameter. Pressure studies on viruses have direct biotechnological applications. Currently, most studies that involve viral inactivation by HHP are found in the area of food engineering and focus on the inactivation of foodborne viruses. Nevertheless, studies of viral inactivation for other purposes have also been conducted. HHP has been shown to be efficient in the inactivation of many viruses of clinical importance and the use of HHP approach has been proposed for the development of animal and human vaccines. Several studies have demonstrated that pressure can result in virus inactivation while preserving immunogenic properties. Viruses contain several components that can be susceptible to the effects of pressure. HHP has been a valuable tool for assessing viral structure function relationships because the viral structure is highly dependent on protein-protein interactions. In the case of small icosahedral viruses, incremental increases in pressure produce a progressive decrease in the folding structure when moving from assembled capsids to ribonucleoprotein intermediates (in RNA viruses), free dissociated units (dimers and/or monomers) and denatured monomers. High pressure inactivates enveloped viruses by trapping their particles in a fusion-like intermediate state. The fusogenic state, which is characterized by a smaller viral volume, is the final conformation promoted by HHP, in contrast with the metastable native state, which is characterized by a larger volume. The combined effects of high pressure with other factors, such as low or subzero temperature, pH and agents in sub-denaturing conditions (urea), have been a formidable tool in the assessment of the component's structure, as well as pathogen inactivation. HHP is a technology for the production of inactivated vaccines that are free of chemicals, safe and capable of inducing strong humoral and cellular immune responses. Here we present a current overview about the pressure-induced viral inactivation and the production of inactivated viral vaccines.

Yellow fever vaccine protects mice against Zika virus infection.

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Polyclonal F(ab')2 fragments of equine antibodies raised against the spike protein neutralize SARS-CoV-2 variants with high potency.

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:106, and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT90). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab')2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab')2 to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab')2 was similar against the WT and P.2 variants, but displaced to higher concentrations by 0.39 log units against the P.1 (Gamma) variant. These results support the possibility of using equine F(ab')2 preparation for the clinical treatment of COVID patients.

VP4 protein from human rhinovirus 14 is released by pressure and locked in the capsid by the antiviral compound WIN.

Rhinoviruses are the major causative agents of the common cold in humans. Here, we studied the stability of human rhinovirus type 14 (HRV14) under conditions of high hydrostatic pressure, low temperature, and urea in the absence and presence of an antiviral drug. Capsid dissociation and changes in the protein conformation were monitored by fluorescence spectroscopy, light scattering, circular dichroism, gel filtration chromatography, mass spectrometry and infectivity assays. The data show that high pressure induces the dissociation of HRV14 and that this process is inhibited by WIN 52084. MALDI-TOF mass spectrometry experiments demonstrate that VP4, the most internal viral protein, is released from the capsid by pressure treatment. This release of VP4 is concomitant with loss of infectivity. Our studies also show that at least one antiviral effect of the WIN drugs involves the locking of VP4 inside the capsid by blocking the dynamics associated with cell attachment.

Pressure-inactivated yellow fever 17DD virus: implications for vaccine development.

The successful Yellow Fever (YF) vaccine consists of the live attenuated 17D-204 or 17DD viruses. Despite its excellent record of efficacy and safety, serious adverse events have been recorded and influenced extensive vaccination in endemic areas. Therefore, alternative strategies should be considered, which may include inactivated whole virus. High hydrostatic pressure has been described as a method for viral inactivation and vaccine development. The present study evaluated whether high hydrostatic pressure would inactivate the YF 17DD virus. YF 17DD virus was grown in Vero cells in roller bottle cultures and subjected to 310MPa for 3h at 4 degrees C. This treatment abolished YF infectivity and eliminated the ability of the virus to cause disease in mice. Pressure-inactivated virus elicited low level of neutralizing antibody titers although exhibited complete protection against an otherwise lethal challenge with 17DD virus in the murine model. The data warrant further development of pressure-inactivated vaccine against YF.

On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles.

Mayaro virus (MAYV) is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

Stability of different influenza subtypes: How can high hydrostatic pressure be a useful tool for vaccine development?

BACKGROUND: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. METHODS: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. RESULTS: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. CONCLUSIONS: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.

Pressure induces folding intermediates that are crucial for protein-DNA recognition and virus assembly.

Protein-nucleic acid interactions are crucial for a variety of fundamental biological processes such as replication, transcription, restriction, translation and virus assembly. The molecular basis of protein-DNA and protein-RNA recognition is deeply related to the thermodynamics of the systems. We review here how protein-nucleic acid interactions can be approached in the same way as protein-protein interactions involved in protein folding and protein assembly, using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states or molten-globule states of a protein. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. The pressure studies reveal that a gradient of partially folded (molten globule) conformations is present between the unfolded and fully folded structure of several bacteria, plant and mammalian viruses. Using pressure, we have detected the presence of a ribonucleoprotein intermediate, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Pressure studies on viruses have direct biotechnological applications. The ability of pressure to inactivate viruses has been evaluated with a view toward the applications of vaccine development and virus sterilization. Recent studies demonstrate that pressure causes virus inactivation while preserving the immunogenic properties. There is substantial evidence that a high-pressure cycle traps a virus in the 'fusion intermediate state', not infectious but highly immunogenic.

Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice.

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.

Inhibition of Mayaro virus infection by bovine lactoferrin.

Mayaro virus (MAYV) is an arbovirus linked to several sporadic outbreaks of a highly debilitating febrile illness in many regions of South America. MAYV is on the verge of urbanization from the Amazon region and no effective antiviral intervention is available against human infections. Our aim was to investigate whether bovine lactoferrin (bLf), an iron-binding glycoprotein, could hinder MAYV infection. We show that bLf promotes a strong inhibition of virus infection with no cytotoxic effects. Monitoring the effect of bLf on different stages of infection, we observed that virus entry into the cell is the heavily compromised event. Moreover, we found that binding of bLf to the cell is highly dependent on the sulfation of glycosaminoglycans, suggesting that bLf impairs virus entry by blocking these molecules. Our findings highlight the antiviral potential of bLf and reveal an effective strategy against one of the major emerging human pathogens in the neotropics.

Full inactivation of human influenza virus by high hydrostatic pressure preserves virus structure and membrane fusion while conferring protection to mice against infection.

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.

The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

Effects of hydrostatic pressure on the stability and thermostability of poliovirus: a new method for vaccine preservation.

Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1-3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 degrees C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use.

Conformational changes in bovine lactoferrin induced by slow or fast temperature increases.

Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70 degrees C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25 degrees C to 105 degrees C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.

The proapoptotic protein Smac/DIABLO dimer has the highest stability as measured by pressure and urea denaturation.

Apoptosis is an essential mechanism of cell death required for normal development and homeostasis of all multicellular organisms. Smac/DIABLO is a dimeric protein important in the control of apoptosis by removing the inhibitory activity of IAPs (inhibitor of apoptosis proteins). In vitro studies reveal that dimerization is required for its function. Here we investigate the structural and thermodynamic features of folding and dimerization of Smac/DIABLO. To disturb the folded, dimeric structure, we used high hydrostatic pressure, low and high temperatures, and chemical denaturing agents. Conformational changes were monitored using spectroscopic techniques such as fluorescence and circular dichroism (CD) as well as gel filtration chromatography. Our data show that Smac/DIABLO is very stable under pressures up to 3.1 kbar, even at subzero temperatures. A complete denaturation/dissociation process is obtained when we use high concentrations of urea, which affect its secondary structure as assessed by CD. The association of pressure and subdenaturing urea concentrations also results in complete denaturation/dissociation of the protein. Under these conditions, unfolding of the protein shows concentration dependence that is in accordance with the dimer-monomer dissociation equilibrium, confirming Smac/DIABLO dissociation. These results suggest that most of the treatments lead to a reversible disruption of the dimeric structure with a dissociation constant ( K d) of 34 x 10 (-21) M (34 zM). This tight dimer is biologically relevant, considering that monomeric mutants bind IAP with low affinity. The extremely high stability of the dimeric form of Smac/DIABLO also implies that once expressed in the cell the protein has a low probability of dissociation and, consequently, loss of function. In addition, the stability in the zeptomolar range is the highest so far measured for a dimeric protein. It also indicates that under most circumstances Smac/DIABLO does not exist as a monomer in the cell and suggests that the dimer-to-monomer equilibrium does not play a regulatory role in the Smac/DIABLO-IAP interaction.

Structure of a membrane-binding domain from a non-enveloped animal virus: insights into the mechanism of membrane permeability and cellular entry.

The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses.