Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.

Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound.

OBJECTIVE: Surgical infusion of gene therapy vectors has provided opportunities for biological manipulation of specific brain circuits in both animal models and human patients. Transient focal opening of the blood-brain barrier (BBB) by MR-guided focused ultrasound (MRgFUS) raises the possibility of noninvasive CNS gene therapy to target precise brain regions. However, variable efficiency and short follow-up of studies to date, along with recent suggestions of the potential for immune reactions following MRgFUS BBB disruption, all raise questions regarding the viability of this approach for clinical translation. The objective of the current study was to evaluate the efficiency, safety, and long-term stability of MRgFUS-mediated noninvasive gene therapy in the mammalian brain. METHODS: Focused ultrasound under the control of MRI, in combination with microbubbles consisting of albumin-coated gas microspheres, was applied to rat striatum, followed by intravenous infusion of an adeno-associated virus serotype 1/2 (AAV1/2) vector expressing green fluorescent protein (GFP) as a marker. Following recovery, animals were followed from several hours up to 15 months. Immunostaining for GFP quantified transduction efficiency and stability of expression. Quantification of neuronal markers was used to determine histological safety over time, while inflammatory markers were examined for evidence of immune responses. RESULTS: Transitory disruption of the BBB by MRgFUS resulted in efficient delivery of the AAV1/2 vector to the targeted rodent striatum, with 50%-75% of striatal neurons transduced on average. GFP transgene expression appeared to be stable over extended periods of time, from 2 weeks to 6 months, with evidence of ongoing stable expression as long as 16 months in a smaller cohort of animals. No evidence of substantial toxicity, tissue injury, or neuronal loss was observed. While transient inflammation from BBB disruption alone was noted for the first few days, consistent with prior observations, no evidence of brain inflammation was observed from 2 weeks to 6 months following MRgFUS BBB opening, despite delivery of a virus and expression of a foreign protein in target neurons. CONCLUSIONS: This study demonstrates that transitory BBB disruption using MRgFUS can be a safe and efficient method for site-specific delivery of viral vectors to the brain, raising the potential for noninvasive focal human gene therapy for neurological disorders.

Electron tomography imaging of surface glycoproteins on human parainfluenza virus 3: association of receptor binding and fusion proteins before receptor engagement.

UNLABELLED: In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a "heads-down" configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended "heads-up" form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN's globular head does HN transmit its activating signal to F. IMPORTANCE: Human parainfluenza virus 3 (HPIV3) is an enveloped, ssRNA virus that can cause serious respiratory illness, especially in children. HPIV3, like most other paramyxoviruses, uses two specialized proteins to mediate cell entry: the fusion protein (F) and the receptor binding protein, hemagglutinin-neuraminidase (HN). F becomes activated to mediate fusion during entry when it is triggered by a signal from HN. Here we used electron tomography to reconstruct the 3-dimensional ultrastructure of HPIV3. From these structures, we could discern the distribution and, in some cases, conformation of HN and F proteins, which provided an understanding of their interrelationship on virions. HN is found in arrays alone in one conformation and interspersed with prefusion F trimers in another. The data support a model of paramyxovirus membrane fusion in which HN associates with F before receptor engagement, and receptor engagement by the globular head of HN switches the HN-F interaction into one of fusion activation.

Measles fusion machinery is dysregulated in neuropathogenic variants.

UNLABELLED: Paramyxoviruses, including the human pathogen measles virus (MV), enter host cells by fusing their viral envelope with the target cell membrane. This fusion process is driven by the concerted actions of the two viral envelope glycoproteins, the receptor binding protein (hemagglutinin [H]) and the fusion (F) protein. H attaches to specific proteinaceous receptors on host cells; once the receptor engages, H activates F to directly mediate lipid bilayer fusion during entry. In a recent MV outbreak in South Africa, several HIV-positive people died of MV central nervous system (CNS) infection. We analyzed the virus sequences from these patients and found that specific intrahost evolution of the F protein had occurred and resulted in viruses that are "CNS adapted." A mutation in F of the CNS-adapted virus (a leucine-to-tryptophan change present at position 454) allows it to promote fusion with less dependence on engagement of H by the two known wild-type (wt) MV cellular receptors. This F protein is activated independently of H or the receptor and has reduced thermal stability and increased fusion activity compared to those of the corresponding wt F. These functional effects are the result of the single L454W mutation in F. We hypothesize that in the absence of effective cellular immunity, such as HIV infection, MV variants bearing altered fusion machinery that enabled efficient spread in the CNS underwent positive selection. IMPORTANCE: Measles virus has become a concern in the United States and Europe due to recent outbreaks and continues to be a significant global problem. While live immunization is available, there are no effective therapies or prophylactics to combat measles infection in unprotected people. Additionally, vaccination does not adequately protect immunocompromised people, who are vulnerable to the more severe CNS manifestations of disease. We found that strains isolated from patients with measles virus infection of the CNS have fusion properties different from those of strains previously isolated from patients without CNS involvement. Specifically, the viral entry machinery is more active and the virus can spread, even in the absence of H. Our findings are consistent with an intrahost evolution of the fusion machinery that leads to neuropathogenic MV variants.

Unity in diversity: shared mechanism of entry among paramyxoviruses.

The Paramyxoviridae family includes many viruses that are pathogenic in humans, including parainfluenza viruses, measles virus, respiratory syncytial virus, and the emerging zoonotic Henipaviruses. No effective treatments are currently available for these viruses, and there is a need for efficient antiviral therapies. Paramyxoviruses enter the target cell by binding to a cell surface receptor and then fusing the viral envelope with the target cell membrane, allowing the release of the viral genome into the cytoplasm. Blockage of these crucial steps prevents infection and disease. Binding and fusion are driven by two virus-encoded glycoproteins, the receptor-binding protein and the fusion protein, that together form the viral "fusion machinery." The development of efficient antiviral drugs requires a deeper understanding of the mechanism of action of the Paramyxoviridae fusion machinery, which is still controversial. Here, we review recent structural and functional data on these proteins and the current understanding of the mechanism of the paramyxovirus cell entry process.

Regulation of paramyxovirus fusion activation: the hemagglutinin-neuraminidase protein stabilizes the fusion protein in a pretriggered state.

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.