Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na+ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na+ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na+ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

The influenza A virus pandemic of 1918–1919 resulted in an estimated 20–40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Long term trends in the evolution of H(3) HA1 human influenza type A

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain’s epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 × 10−3 substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 ± 5, significantly fewer than in the twigs (90 ± 7), which in turn is significantly fewer variable codons than in tip branches (175 ± 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution

In this paper we determine the extent to which host-mediated mutations and a known sampling bias affect evolutionary studies of human influenza A. Previous phylogenetic reconstruction of influenza A (H3N2) evolution using the hemagglutinin gene revealed an excess of nonsilent substitutions assigned to the terminal branches of the tree. We investigate two hypotheses to explain this observation. The first hypothesis is that the excess reflects mutations that were either not present or were at low frequency in the viral sample isolated from its human host, and that these mutations increased in frequency during passage of the virus in embryonated eggs. A set of 22 codons known to undergo such “host-mediated” mutations showed a significant excess of mutations assigned to branches attaching sequences from egg-cultured (as opposed to cell-cultured) isolates to the tree. Our second hypothesis is that the remaining excess results from sampling bias. Influenza surveillance is purposefully biased toward sequencing antigenically dissimilar strains in an effort to identify new variants that may signal the need to update the vaccine. This bias produces an excess of mutations assigned to terminal branches simply because an isolate with no close relatives is by definition attached to the tree by a relatively long branch. Simulations show that the magnitude of excess mutations we observed in the hemagglutinin tree is consistent with expectations based on our sampling protocol. Sampling bias does not affect inferences about evolution drawn from phylogenetic analyses. However, if possible, the excess caused by host-mediated mutations should be removed from studies of the evolution of influenza viruses as they replicate in their human hosts.

Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

Chronic stress alters the immune response to influenza virus vaccine in older adults.

To determine whether a chronic stressor (caregiving for a spouse with a progressive dementia) is associated with an impaired immune response to influenza virus vaccination, we compared 32 caregivers' vaccine responses with those of 32 sex-, age-, and socioeconomically matched control subjects. Caregivers showed a poorer antibody response following vaccination relative to control subjects as assessed by two independent methods, ELISA and hemagglutination inhibition. Caregivers also had lower levels of in vitro virus-specific-induced interleukin 2 levels and interleukin 1beta; interleukin 6 did not differ between groups. These data demonstrate that down-regulation of the immune response to influenza virus vaccination is associated with a chronic stressor in the elderly. These results could have implications for vulnerability to infection among older adults.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation.

The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable. HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide. They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.