Debi P. Nayak

Assembly and budding of influenza virus

Abstract Influenza viruses are causative agents of an acute febrile respiratory disease called influenza (commonly known as “flu”) and belong to the Orthomyxoviridae family. These viruses possess segmented, negative stranded RNA genomes (vRNA) and are enveloped, usually spherical and bud from the plasma membrane (more specifically, the apical plasma membrane of polarized epithelial cells). Complete virus particles, therefore, are not found inside infected cells. Virus particles consist of three major subviral components, namely the viral envelope, matrix protein (M1), and core (viral ribonucleocapsid [vRNP]). The viral envelope surrounding the vRNP consists of a lipid bilayer containing spikes composed of viral glycoproteins (HA, NA, and M2) on the outer side and M1 on the inner side. Viral lipids, derived from the host plasma membrane, are selectively enriched in cholesterol and glycosphingolipids. M1 forms the bridge between the viral envelope and the core. The viral core consists of helical vRNP containing vRNA (minus strand) and NP along with minor amounts of NEP and polymerase complex (PA, PB1, and PB2). For viral morphogenesis to occur, all three viral components, namely the viral envelope (containing lipids and transmembrane proteins), M1, and the vRNP must be brought to the assembly site, i.e. the apical plasma membrane in polarized epithelial cells. Finally, buds must be formed at the assembly site and virus particles released with the closure of buds. Transmembrane viral proteins are transported to the assembly site on the plasma membrane via the exocytic pathway. Both HA and NA possess apical sorting signals and use lipid rafts for cell surface transport and apical sorting. These lipid rafts are enriched in cholesterol, glycosphingolipids and are relatively resistant to neutral detergent extraction at low temperature. M1 is synthesized on free cytosolic polyribosomes. vRNPs are made inside the host nucleus and are exported into the cytoplasm through the nuclear pore with the help of M1 and NEP. How M1 and vRNPs are directed to the assembly site on the plasma membrane remains unclear. The likely possibilities are that they use a piggy-back mechanism on viral glycoproteins or cytoskeletal elements. Alternatively, they may possess apical determinants or diffuse to the assembly site, or a combination of these pathways. Interactions of M1 with M1, M1 with vRNP, and M1 with HA and NA facilitate concentration of viral components and exclusion of host proteins from the budding site. M1 interacts with the cytoplasmic tail (CT) and transmembrane domain (TMD) of glycoproteins, and thereby functions as a bridge between the viral envelope and vRNP. Lipid rafts function as microdomains for concentrating viral glycoproteins and may serve as a platform for virus budding. Virus bud formation requires membrane bending at the budding site. A combination of factors including concentration of and interaction among viral components, increased viscosity and asymmetry of the lipid bilayer of the lipid raft as well as pulling and pushing forces of viral and host components are likely to cause outward curvature of the plasma membrane at the assembly site leading to bud formation. Eventually, virus release requires completion of the bud due to fusion of the apposing membranes, leading to the closure of the bud, separation of the virus particle from the host plasma membrane and release of the virus particle into the extracellular environment. Among the viral components, M1 contains an L domain motif and plays a critical role in budding. Bud completion requires not only viral components but also host components. However, how host components facilitate bud completion remains unclear. In addition to bud completion, influenza virus requires NA to release virus particles from sialic acid residues on the cell surface and spread from cell to cell. Elucidation of both viral and host factors involved in viral morphogenesis and budding may lead to the development of drugs interfering with the steps of viral morphogenesis and in disease progression.

High-Density Lipoprotein Loses Its Anti-Inflammatory Properties During Acute Influenza A Infection

BackgroundViruses have been identified as one of a variety of potential agents that are implicated in atherogenesis. Methods and ResultsC57BL/6J mice were killed before or 2, 3, 5, 7, or 9 days after intranasal infection with 105 plaque-forming units (pfu) of Influenza A strain WSN/33. Peak infectivity in lungs was reached by 72 hours, and it returned to baseline by 9 days. No viremia was observed at any time. The activities of paraoxonase and platelet-activating factor acetylhydrolase in HDL decreased after infection and reached their lowest levels 7 days after inoculation. The ability of HDL from infected mice to inhibit LDL oxidation and LDL-induced monocyte chemotactic activity in human artery wall cell cocultures decreased with time after inoculation. Moreover, as the infection progressed, LDL more readily induced monocyte chemotaxis. Peak interleukin-6 and serum amyloid A plasma levels were observed at 2 and 7 days after inoculation. HDL apoA-I levels did not change. ApoJ and ceruloplasmin levels in HDL peaked 3 days after infection. Ceruloplasmin remained elevated throughout the time course, whereas apoJ levels decreased toward baseline after the third day. ConclusionsWe conclude that alterations in the relative levels of paraoxonase, platelet-activating factor acetylhydrolase, ceruloplasmin, and apoJ in HDL occur during acute influenza infection, causing HDL to lose its anti-inflammatory properties.

Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein

ABSTRACT Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.

Influenza virus morphogenesis and budding

Abstract Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major sub-viral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins. Budding processes involve bud initiation, bud growth and bud release. The presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching-off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.

Influenza Infection Promotes Macrophage Traffic Into Arteries of Mice That Is Prevented by D-4F, an Apolipoprotein A-I Mimetic Peptide

Background—We reported that HDL loses its antiinflammatory properties during acute influenza A infection in mice, and we hypothesized that these changes might be associated with increased trafficking of macrophages into the artery wall. The present study tested this hypothesis. Methods and Results—D-4F, an apolipoprotein A-I mimetic peptide, or vehicle in which it was dissolved (PBS) was administered daily to LDL receptor–null mice after a Western diet and after influenza infection. D-4F treatment increased plasma HDL cholesterol and paraoxonase activity compared with PBS and inhibited increases in LDL cholesterol and peak levels of interleukin-6 after infection. Lung viral titers were reduced by 50% in mice receiving D-4F. Injection of female mice with male macrophages, which were detected with real-time polymerase chain reaction to measure the male Sry gene, revealed a marked increase in macrophage traffic into the aortic arch and innominate arteries after infection that was prevented by administration of D-4F. Conclusions—We conclude that loss of antiinflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.

Analysis of the Transmembrane Domain of Influenza Virus Neuraminidase, a Type II Transmembrane Glycoprotein, for Apical Sorting and Raft Association

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508–6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH2 terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH2 terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses

Influenza virus assembly and morphogenesis require transport of viral components to the assembly site at the apical plasma membrane of polarized epithelial cells and interaction among the viral components. In this report we have discussed the apical determinants present in the transmembrane domain (TMD) of influenza virus hemagglutinin (HA) and neuraminidase (NA), and the interaction of M1 with influenza virus HA and NA. Earlier studies have shown that the NA and HA TMDs possess determinant(s) for apical sorting and raft-association (Kundu et al., 1996. J. Virol 70, 6508-6515; Lin et al., 1998. J. Cell Biol. 142, 51-57). Analysis of chimeric constructs between NA and TR (human transferring receptor) TMDs and the mutations in the NA and HA TMD sequences showed that the COOH terminus of the NA TMD and NH(2) terminus of the HA TMD encompassing the exoplasmic leaflet of the lipid bilayers were significantly involved in lipid raft-association and that apical determinants were not discrete sequences but rather dispersed within the TMD of HA and NA. These analyses also showed that although both signals for apical sorting and raft-association resided in the NA TMD, they were not identical and varied independently. Interactions of M1 protein with HA or NA, the influenza virus envelope glycoproteins, were investigated by TX-100 detergent treatment of membrane fractions and floatation in sucrose gradients. Results from these analyses showed that the interaction of M1 with mature HA and NA, which associated with the detergent-resistant lipid rafts caused an increased detergent-resistance of the membrane-bound M1 and that M1 interacted with HA and NA both in influenza virus-infected cells as well as in recombinant vaccinia virus-infected cells coexpressing M1 with HA and/or NA. Furthermore, both the cytoplasmic tail and the TMD of HA caused an increased detergent-resistance of the membrane-bound M1 supporting their interaction with M1. Immunofluorescence analysis by confocal microscopy also showed colocalization supporting the interaction of M1 with HA and NA at the cell surface and during exocytic transport both in influenza virus-infected cells as well as in coexpressing cells.

Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding.

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.

Defective-Interfering (DI) RNAs of Influenza Viruses: Origin, Structure, Expression, and Interference

Influenza virus was the first virus for which defective-interfering particles were reported. Von Magnus (1947) observed that when he passaged influenza viruses serially undiluted in embryonated chicken eggs, both the amount of total virus particles as assayed by hemagglutinating units (HAU) and the amount of infectious virus particles as assayed by egg infectivity titer (EID50) decreased. However, he observed that the proportion of infectious particles as assayed by EID50 decreased precipitously when compared to the total virus particle (HAU) production. During the undiluted passages many particles were produced which were noninfectious. These noninfectious influenza virus particles produced at high multiplicity were called “incomplete” particles. Von Magnus (1951 a–c) also described the phenomenon of interference when he reported that these “incomplete” particles interfered with the multiplication of complete (infectious, nondefective, or standard) virus particles. This phenomenon of multiplicity dependent production of noninfectious virus particles is also called the “von Magnus phenomenon.” Since this observation was made with influenza viruses, similar multiplicity-dependent formation of noninfectious particles has been observed with almost all animal viruses carefully studied to date; indeed, formation of such particles has also been reported for plant, yeast, and bacterial viruses (Kane et al. 1979; Mills et al. 1967) and, thus, probably represents a general phenomenon for all viruses. The term “defective-interfering” (DI) particles was introduced by Huang and Baltimore (1970) to describe the definitive characteristics of these noninfectious particles and to separate them from other noninfectious particles which may be noninterfering.