Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Abstract

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 1C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 1C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.Membranes of most enveloped viruses form by budding out from the plasma membranes of their host cells a highly select subset of plasma membrane components. In general, the selected membrane proteins are coded by the viral genome, whereas lipids are recruited from host membranes; however, the lipid composition of the viral envelope differs from that of the host membrane 1,2 and from other budding viruses 3 . The envelope of influenza contains higher amounts of both cholesterol 1 and glycosphingolipids 4 -lipids known to partition into the liquid ordered (l o ) phase. The l o phase is characterized by extended hydrocarbon chains having a reduced gauche-trans isomerization compared with those of the liquid disordered (l d ) phase, but having a similar rotational and translational mobility 5 . Liquid ordered phases are thought to be at the core of lipid rafts 6 , which are defined as transient membrane microdomains that are enriched in sphingolipids and cholesterol (1) 7 -a hypothesis that has generated much debate 8,9 .The influenza virus has played a pivotal role in the development of the raft hypothesis, starting with early studies that inferred ordered domains using spin probes 10-12 and fluorescence 13,14 and that suggested that an ordered lipid domain is selected in toto as the envelope during budding from the plasma membrane 15 . These lipids are either selected at the time of budding or pre-selected as the 'pre-envelope' suggested by clusters of the viral envelope protein hemagglutinin seen in immunoelectron microscopy 16 .Although detection of virus-sized domains of lipids (B100-nm diameter) is below the limit of resolution of fluorescent microscopy, large micrometer-sized membrane domains are reliably detected [17][18][19] . Recently, proton magic angle spinning NMR ( 1 H MAS NMR) has been used to determine the phase diagram of the same lipid compositions used to study large membrane domains in lipid bilayers and othe...

Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses

“…We next tested whether interactions with the lipid envelope were required for inhibition of infectivity. Most eukaryotic lipid bilayers, including the envelopes of influenza virus (38), are rigid at 4°C. We reasoned that such rigidity would prevent the insertion of hydrophobic compounds into envelopes (but not potential interactions with exposed glycoproteins).…”

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

“…We also briefly investigated the hypothesis that favorable interactions between the influenza hemagglutinin clusters and sphingolipids in the plasma membrane (10)(11)(12) induced the sphingolipid domains we observed. As a first test for cohesive sphingolipid-hemagglutinin interactions, we used fluorescence microscopy to assess whether BODIPY-sphingolipids and immunolabeled hemagglutinin colocalized in the membranes of Clone 15 cells.…”

“…We focused on sphingolipids because (i) they are postulated to be segregated within compositionally distinct lipid domains, such as lipid rafts (10)(11)(12), and (ii) sphingolipid metabolites are signaling molecules that regulate cell survival and proliferation (13). We studied a transfected mouse fibroblast cell line that stably expresses influenza hemagglutinin (Clone 15 cell line) because the hemagglutinin clusters in their plasma membranes are thought to be associated with sphingolipid and cholesterol domains (10)(11)(12). Thus, we hoped to increase the probability of detecting sphingolipid-enriched plasma membrane domains by using the Clone 15 cell line.…”

Lipidomics identifies a requirement for peroxisomal function during influenza virus replication

“…Host cell lipid metabolism and plasma membrane microdomains are implicated in the biogenesis of virus envelopes. Several studies have dissected the lipid inventory of purifi ed infl uenza virions ( 9,10 ), whereas others have demonstrated the requirements for de novo fatty acid and sphingolipid biosynthesis and unique cholesterol compositions for virus production at budding sites (11)(12)(13)(14).…”