Identification of defects in the neuraminidase gene of four temperature-sensitive mutants of A/WSN/33 influenza virus

Four influenza (A/WSN/33) mutants, temperature sensitive (ts) for neuraminidase (NA) (Sugiura et al., 1972, 1975) were analyzed. All four ts mutants were found to be defective at the nonpermissive temperature (39.5 degrees) both in enzymatic activity and in transport to the cell surface. Upon shift down to the permissive temperature (33 degrees), enzymatic activity and transport to the cell surface were both restored suggesting that the mutational defect is reversible. Comparative sequence analysis of the NA gene from ts mutants, their revertants and wild type WSN viruses revealed that in each case single point mutations causing amino acid substitutions were associated with the ts defect. The positions of each point mutation when mapped in the three-dimensional structure of NA varied. However, all four amino acid substitutions were located in beta-sheet strands of the head region. Several other amino acid changes not essential for the ts phenotype were found in each mutant NA. The nonessential changes were localized either in the stalk region or in the loop structures of the head, but none in the beta-sheet strands. Because both enzymatic activity and transport of NA were affected in all four mutants, we propose that the mutational phenotype is caused by a change in overall conformation rather than a localized change in the sialic acid binding site.     

Characterization of a temperature-sensitive mutant in the RNA polymerase PB2 subunit gene of influenza A/WSN/33 virus

Summary The temperature-sensitive mutant ts-1 of influenza virus A/WSN/33 carries mutations in the gene encoding RNA polymerase PB2 subunit. Effect of temperature on various steps of viral RNA synthesis was examined using disrupted virions of ts-1 mutant. The initiation of RNA synthesis with dinucleotide ApG primer was not affected by elevated temperature, whereas that with primer RNA containing 5-terminal cap-1 structure was temperature-sensitive. The result supports the previous notion deduced from the UV-crosslinking experiments, that PB2 is involved in the cap-1 dependent initiation of RNA synthesis. In addition, the ts-1 mutant showed a defect in RNA chain elongation. Nucleotide sequence analysis of RNA segment 1 of ts-1 mutant revealed that the amino acid number 417 is essential for the recognition of cap-1 structures and/or the interaction with catalytic unit of the RNA polymerase.     

Changes in the hemagglutinin gene of the neurovirulent influenza virus strain A/NWS/33

The neurovirulent strain of influenza A virus, A/NWS/33, is able to infect a large range of cell types, including mouse brain cells, which are not infected by its parent, A/WS/33. This seems to be largely due to the hemagglutinin of A/NWS/33. The complete nucleotide sequence of the HA genes of both strains has been determined and a comparison revealed a number of changes. Analysis showed that the virulence capabilities of the NWS HA involve at least three different mechanisms: (a) loss of a glycosylation site; (b) a change at the cleavage site; and (c) a substitution in HA₂, which may increase the pH of fusion.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers U08903-4.     

Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Invasion and persistence of the neuroadapted influenza virus A/WSN/33 in the mouse olfactory system

Invasion and persistence of the neuroadapted influenza virus A/WSN/33 in the mouse olfactory system was studied. WSN/33 instilled intranasally infected neurons in the olfactory epithelium and was transported in axons to the olfactory bulbs in wild type mice that survived the infection. In adult mice lacking the recombination activating gene 1 (RAG-1-/-), infected neurons occurred in the olfactory bulbs for 22-65 days after which the mice developed a rapidly progressive lethal infection affecting neurons in olfactory projection pathways, i.e. primary olfactory cortex, raphe in upper brainstem and hypothalamus. Adult mice without genes for interferon (IFN)-alpha/beta receptor, IFN-gamma receptor, inducible nitric oxide synthase (iNOS), IgH, the transporter associated with antigen processing 1 (TAP1), and natural killer cell-depleted mice, all survived the infection. Viral RNA was found in the olfactory bulbs in more than 80 per cent of the surviving iNOS-/-, IFN-gamma receptor-/-, and TAP1-/- mice. Taken together, this study shows that influenza A virus can invade the brain through the olfactory pathways and that the cellular immune responses prevent establishment of persistent infections in the olfactory bulbs. Furthermore, innate responses in olfactory bulbs may for a period of time keep the infection under control.     

A mutant of influenza virus with a temperature-sensitive defect in the posttranslational processing of the hemagglutinin.

A mutant of fowl plague virus (ts 227) with a temperature-sensitive defect in the hemagglutinin [Scholtissek, C., and Bowles, A. L. (1975). Virology67, 576–587] has been analyzed. At the nonpermissive temperature, the hemagglutinin is synthesized and incorporated into the rough endoplasmic reticulum, but is not transported to the smooth endoplasmic reticulum and to the cell surface. As a result of this defect in migration, proteolytic cleavage of the hemagglutinin does not occur and glycosylation is incomplete, in that mannose and glucosamine are incorporated into the glycoprotein, whereas galactose and fucose are attached only at the permissive temperature. Another step involved in glycosylation, which occurs also only at the permissive temperature, is partial removal of mannose in a trimming process. The data suggest that the hemagglutinin has a structural feature which, after insertion of the molecule into the rough endoplasmic reticulum, promotes its transport to the cell surface. The hemagglutinin incorporated into the rough endoplasmic reticulum at the nonpermissive temperature does not show hemagglutinating activity. After solubilization with detergent, it reacts with antiserum specified against wild-type hemagglutinin. When migration of the hemagglutinin is blocked, synthesis of the neuraminidase proceeds normally, indicating that there is little interdependence in the processing of both envelope glycoproteins.     

Mutation in the hemagglutinin of A/N-WS/33 influenza virus recombinants influencing sensitivity to trypsin and antigenic reactivity

The hemagglutination activity of the influenza virus A/RI/5-/57 (H2N2) is resistant to trypsin treatment, but that of A/N-WS/33 (HONI) is rapidly lost on exposure to trypsin. Two recombinant viruses, both of which derive their HO hemagglutinin from NWS and their N2 neuraminidase from RI/5^?, show different sensitivity to trypsin. The hemagglutination activity of X-12, like that of NWS, is rapidly lost, whereas the activity of X-29 shows a trypsin sensitivity intermediate between that of the NWS and RI/5^? parental viruses. Trypsin sensitivity of hemagglutination activity is a phenotypic marker that is associated in the present case with minimal antigenic differences as revealed by conventional serological techniques using subtype-specific antisera. The specific property of slow trypsin sensitivity, which can revert to fast trypsin sensitivity, is probably due to one or a limited number of point mutations in the hemagglutinin gene. The selection of minor antigenic variants may occur fortuitously during exposure of the virus to nonimmunologic pressures. Although X-12 and X-29 viruses differ with respect to ratios of hemagglutinin and neuraminidase proteins per virion, neither the amount nor the antigenic nature of the neuraminidase protein influences the relative trypsin sensitivity of the associated viral hemagglutinin. Selective removal of hemagglutinin spikes by trypsin has revealed the morphology and spacing of the neuraminidase spikes in situ.     

Sequence of the hemagglutinin gene from influenza virus A/Seal/Mass/1/80

A double-strand DNA copy of the influenza virus A/Seal/Mass/1/80 (H7N7) [seal] hemagglutinin (HA) gene was cloned into the plasmid pAT153/PvuII/8 and sequenced to deduce the primary amino acid sequence. The gene is 1731 nucleotides long and codes for a protein of 560 amino acids with a nonglycosylated molecular weight of 62098 Da. The deduced amino acid sequence displays similarities to all other sequenced hemagglutinins by retaining six of seven potential glycosylation sites, showing conversation in the number and position of cysteine residues, conservation in the fusion and anchor peptides, and conservation in the putative receptor site of the molecule. However, three features of the primary amino acid sequence could be distinguished from the H7 amino acid sequence of A/fowl plague/Rostock/34 (FPV), another avian H7 influenza virus which does not produce disease in mammals. First, the seal HA sequence has three fewer amino acids in the connecting peptide region of the HA than FPV. This lack of multiple basic amino acids in the connecting peptide is similar to that found in avirulent H7 avian strains and to mammalian serotypes H1, H2, and H3. Second, the seal HA has gained four additional proline residues, all in HA1, as compared to FPV. These residues may alter the tertiary structure of the HA and ultimately contribute to the biological features of this virus. Third, the seal HA has lost a potential carbohydrate attachment site at residue 149 which lies at the tip of the HA structure. The loss of this carbohydrate could alter the seal HAs interaction with host cell receptors.     

Further isolation and characterization of temperature-sensitive mutants of influenza virus.

We have assigned 34 temperature-sensitive (ts) mutants of the WSN strain of influenza virus into seven nonoverlapping recombination-complementation groups by conducting pairwise crosses. A single gene of the viral genome was affected in 27 mutants while six were double mutants and one was a triple mutant. The synthesis of virion RNA in mutant-infected cells at the nonpermissive temperature was studied by the incorporation of [³H]uridine in the presence of actinomycin D into an acid-insoluble product. This method separated mutants into two classes. Mutants belonging to Groups I, II, III, and V were RNA^?, while those belonging to Groups IV, VI, and VII were RNA⁺. The results of temperature shift-up experiments suggested that all four groups of RNA^? mutants were defective in replicating virion RNA. Production of serologically or functionally active subviral components (ribonucleoprotein, hemagglutinin, and neuraminidase) by RNA^? mutants at the nonpermissive temperature was variable. RNA⁺ mutants produced a normal amount of these components, except Group IV mutants in which the production of functional hemagglutinin and neuraminidase was greatly depressed.     

Temperature-sensitive mutants of influenza A virus: Evaluation of the Alaska/77-ts-1 A 2 temperature-sensitive recombinant virus in seronegative adult volunteers

Summary An influenza A virus recombinant bearing the surface antigens of the A/Alaska/6/77 (H 3 N 2) wild type virus and the twots genes of the A/Udorn/72-ts-1 A 2 (H 3 N 2) virus was evaluated for attenuation, antigenicity, and transmissibility in 28 adult volunteers all of whom possessed a preinoculation serum hemagglutination-inhibiting (HAI) antibody titer of 1:8 and 18 of whom also possessed a serum neuraminidase-inhibiting (NI) antibody titer of 1:4. The Alaska/77-ts-1 A 2 recombinant, which had a 37° C shutoff temperature for plaque formation andts mutations on the genes thought to code for the P1 and P3 polymerase proteins, infected 71 percent of the vaccinees when administered at a dose of 10^6.5 TCID₅₀. Only 3 percent of the vaccinees developed symptoms in contrast to 50 percent of volunteers who received 10^4.2 TCID₅₀ of wild type virus. Vaccinees shed virus for a shorter interval and at a lower titer than the volunteers who received wild type virus. Eachts-1 A 2 isolate retained thets phenotype indicating that the recombinant was stable genetically in seronegative adults. An immunological response, as measured by a rise in serum HAI and/or NI antibody, was detected in 71 percent of the vaccinees and 87 percent of the recipients of wild type virus. Transmission of vaccine virus to susceptible contacts was not observed. The twots-1 A 2ts genes have now been transferred to two variants within the H 3 N 2 subtype, the Vic/75 and Alaska/77 viruses, and have rendered the viruses satisfactorily attenuated for adults. The level of infectivity of the Alaska/77-ts-1 A 2 virus appeared to be low, however.This work supported in part by contracts N01-A 1-42553 and N01-A1-22503 sponsored by the National Institute of Allergy and Infectious Diseases.     

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.     

Studies of two temperature-sensitive mutants of Moloney murine leukemia virus

Events associated with the replication of two spontaneous temperature-sensitive mutants (ts1 and ts3) of Moloney murine leukemia virus were studied under non-permissive and permissive conditions of infection. From temperature-shift experiments ts1 appears to have a temperature-sensitive step occurring early in the growth cycle and in an unidentified function. The temperature-sensitive defect of ts3 occurs late in the replicative cycle. Electron microscope studies showed that although budding partially occurs at the nonpermissive temperature there is no release of virus particles from the cell membrane. The defect of ts3 was also found to be in one of the factors required for the rescue of MuSV.     

Temperature-sensitive mutants of influenza A virus: evaluation of the A/Victoria/75-ts-1A2 temperature-sensitive recombinant virus in seronegative adult volunteers.

An influenza A virus recombinant bearing the surface antigens of the A/Victoria/3/75 (H3N2) strain and the two ts genes of the A/Udorn/72-ts-1A2 virus was evaluated for attenuation, antigenicity, and protective effect in 42 doubly seronegative adult volunteers (i.e., individuals who lacked detectable serum antibodies for the hemagglutinin and neuraminidase antigens). This recombinant, which had a 37 degrees C shutoff temperature for plaque formation and ts mutations on the genes thought to code for the P1 and P3 polymerase proteins, infected 90% of the volunteers. Of the volunteers, 5% developed mild coryza or rhinitis but other signs or symptoms were not observed, indicating that the A/Victoria/75-ts-1A2 recombinant was more attenuated than the A/Victoria/75-ts-1[E] recombinant. Vaccinees shed virus for a shorter interval and at a lower titer than did the A/Victoria/75-ts-1[E] vaccinees. Each ts-1A2 isolate retained the ts phenotype indicating that the recombinant was stable genetically in doubly seronegative adults. Finally, the ts-1A2 recombinant induced significant resistance to subsequent challenge with A/Victoria/75 wild-type virus.