Human parainfluenza virus type 1 regulates cholesterol biosynthesis and establishes quiescent infection in human airway cells.

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

Specificity and functional interplay between influenza virus PA-X and NS1 shutoff activity.

Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed "host shutoff." Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We analyzed viral growth, general shutoff activity, specificity of mRNA targets, and viral gene expressions. Our results showed that PA-X was the major contributor in reducing general host protein expression in the virus-infected cells. Intriguingly, our transcriptomic analysis from infected human airway A549 cells indicate that shutoff-active NS1 specifically targeted host mRNAs related to interferon (IFN) signaling pathways and cytokine release. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of shutoff-active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing shutoff-active NS1 with reduced amount of PA-X expression most efficiently suppressed antiviral and innate immune responses in human cells, indicating that influenza virus needs to optimize the contribution of these two shutoff proteins to circumvent host responses for its optimum growth.

Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.

KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.

Activating FcγRs on monocytes are necessary for optimal Mayaro virus clearance.

Mayaro virus (MAYV) is an emerging arbovirus. Previous studies have shown antibody Fc effector functions are critical for optimal monoclonal antibody-mediated protection against alphaviruses; however, the requirement of Fc gamma receptors (FcγRs) for protection during natural infection has not been evaluated. Here, we showed mice lacking activating FcγRs (FcRγ-/-) developed prolonged clinical disease with more virus in joint-associated tissues. Viral clearance was associated with anti-MAYV cell surface binding rather than neutralizing antibodies. Lack of Fc-FcγR engagement increased the number of monocytes through chronic timepoints. Single cell RNA sequencing showed elevated levels of pro-inflammatory monocytes in joint-associated tissue with increased MAYV RNA present in FcRγ-/- monocytes and macrophages. Transfer of FcRγ-/- monocytes into wild type animals was sufficient to increase virus in joint-associated tissue. Overall, this study suggests that engagement of antibody Fc with activating FcγRs promotes protective responses during MAYV infection and prevents monocytes from being potential targets of infection.

DEER EPR measurements for membrane protein structures via bifunctional spin labels and lipodisq nanoparticles.

Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

Impact of Influenza A Virus Shutoff Proteins on Host Immune Responses.

Influenza A virus (IAV) is a significant human pathogen that causes seasonal epidemics. Although various types of vaccines are available, IAVs still circulate among human populations, possibly due to their ability to circumvent host immune responses. IAV expresses two host shutoff proteins, PA-X and NS1, which antagonize the host innate immune response. By transcriptomic analysis, we previously showed that PA-X is a major contributor for general shutoff, while shutoff active NS1 specifically inhibits the expression of host cytokines, MHC molecules, and genes involved in innate immunity in cultured human cells. So far, the impact of these shutoff proteins in the acquired immune response in vivo has not been determined in detail. In this study, we analyzed the effects of PA-X and NS1 shutoff activities on immune response using recombinant influenza A/California/04/2009 viruses containing mutations affecting the expression of shutoff active PA-X and NS1 in a mouse model. Our data indicate that the virus without shutoff activities induced the strongest T and B cell responses. Both PA-X and NS1 reduced host immune responses, but shutoff active NS1 most effectively suppressed lymphocyte migration to the lungs, antibody production, and the generation of IAV specific CD4+ and CD8+ T cells. NS1 also prevented the generation of protective immunity against a heterologous virus challenge. These data indicate that shutoff active NS1 plays a major role in suppressing host immune responses against IAV infection.

Key Role of the Influenza A Virus PA Gene Segment in the Emergence of Pandemic Viruses.

Influenza A viruses (IAVs) are a significant human pathogen that cause seasonal epidemics and occasional pandemics. Avian waterfowl are the natural reservoir of IAVs, but a wide range of species can serve as hosts. Most IAV strains are adapted to one host species and avian strains of IAV replicate poorly in most mammalian hosts. Importantly, IAV polymerases from avian strains function poorly in mammalian cells but host adaptive mutations can restore activity. The 2009 pandemic H1N1 (H1N1pdm09) virus acquired multiple mutations in the PA gene that activated polymerase activity in mammalian cells, even in the absence of previously identified host adaptive mutations in other polymerase genes. These mutations in PA localize within different regions of the protein suggesting multiple mechanisms exist to activate polymerase activity. Additionally, an immunomodulatory protein, PA-X, is expressed from the PA gene segment. PA-X expression is conserved amongst many IAV strains but activity varies between viruses specific for different hosts, suggesting that PA-X also plays a role in host adaptation. Here, we review the role of PA in the emergence of currently circulating H1N1pdm09 viruses and the most recent studies of host adaptive mutations in the PA gene that modulate polymerase activity and PA-X function.

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (Tm) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the Tm titration experiment indicated an increase in Tm values with the addition of SMA polymer and approaches ∼2 µs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.