Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Abstract

Influenza virus assembles and buds at the plasma membrane of virusinfected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Influenza A virus surface proteins are organized to help penetrate host mucus

“…We first sought to characterize the organization and dynamics of proteins in the viral membrane. By labeling HA and NA, along with the viral nucleoprotein, NP, we are able to measure features of virus organization on intact, infectious particles that corroborate and extend previous observations made using electron microscopy (Calder et al, 2010;Chlanda et al, 2015;Harris et 35 al., 2006;Leser and Lamb, 2017). Images of viruses with labeled nucleoprotein (NP, the most abundant protein in vRNP complexes and a proxy for the virus genome) reveal individual foci of NP that localize to one of the virus's poles, which we use as a fiducial mark for comparing HA and NA localization along the viral envelope.…”

“…However, whether or not virus morphology contributes directly to virus transmission -and if so, how -remains unclear. Similarly, although the two major envelope proteins of IAV, HA and NA, have been observed by electron microscopy to cluster non-uniformly on both the viral and pre- 15 viral envelope (Calder et al, 2010;Harris et al, 2006;Leser and Lamb, 2017), whether and how the spatial organization of HA and NA affects virus transmission remains unclear.…”

Influenza A matrix protein M1 is sufficient to induce lipid membrane deformation

“…In the current work we have investigated for the first time the interaction between IAV M1 and free-standing model membranes containing different amounts of PS. The protein concentration (much larger than the reported K d values (8,33)) was chosen so to take into account that, in vivo, viral proteins might be recruited in small PM domains (40) and, therefore, reach a high local concentration (41). Previous investigations on solid-supported bilayers have demonstrated that M1 binds to lipid membranes containing negatively-charged lipids and that M1-lipid binding is accompanied by extensive protein multimerization (7).…”

Entropic Forces Drive Clustering and Spatial Localization of Influenza A M2 During Viral Budding

“…Other evidence contradicts the idea M2 is associating with nano-domains (or socalled "rafts") (31) and that the CRAC sequence plays a role herein (32). Nonetheless, the presence of a molecular specificity pocket on M2 toward cholesterol might provide general affinity toward the budozone area (33) or in the later stage of budding (34). While structural modulators of M2 (such as cholesterol) might affect the CG model if for instance cholesterolbinding activity can change the affinity of M2 towards the Lo budozone, it appears that M2 recruitment to the budozone is due to interactions with the matrix protein and not due to the cholesterol-binding activities.…”