Identification of the defective genes in three mutant groups of influenza virus.

Seven complementation-recombination groups of temperature-sensitive (ts) influenza WSN virus mutants have been previously isolated. Recently two of these groups (IV and VI) were shown to possess defects in the neuraminidase and the hemagglutinin gene, respectively, and two groups (I and III) were reported to have defects in the P3 and P1 proteins which are required for complementary RNA synthesis. In this communication we report on the defects in the remaining three mutant groups. Wild-type (ts+) recombinants derived from ts mutants and different non-ts influenza viruses were analyzed on RNA polyacrylamide gels. This technique permitted the identification of the P2 protein, the nucleoprotein, and the M protein as the defective gene products in mutant groups II, V, and VII, respectively. Based on the physiological behavior of mutants in groups II and V, it appears that P2 protein and nucleoprotein are required for virion RNA synthesis during influenza virus replication.     

A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase promotes the generation of defective interfering RNAs

An R638A mutation of the polymerase acidic protein (PA) subunit of the RNA polymerase of influenza A/WSN/33 virus results in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering RNAs. We propose that R638A is an "elongation" mutant that destabilizes PA-RNA template interactions during elongation. A C453R mutation in PA can compensate for this defect, suggesting that amino acids C453 and R638 form part of the same domain.     

Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis.

Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees C and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.     

Role of neuraminidase in the morphogenesis of influenza B virus.

When ts7, a temperature-sensitive (ts) mutant of influenza B/Kanagawa/73 virus, infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating activity and enzymatic activity of neuraminidase (NA) were negligible. However, viral protein synthesis and transport of hemadsorption-active hemagglutinin to the cell surface were not affected. When the cell lysate was treated with bacterial NA, hemagglutinating activity was recovered but infectivity was not, even after further treatment with trypsin. It was found that ts7 was defective in transport of NA to the cell surface and formation of virus particles. Analysis of the genomes of non-ts recombinants obtained by crossing ts7 and UV-inactivated B/Lee showed that ts7 had the ts mutation only in RNA segment 6 coding for NA and the glycoprotein NB. Nucleotide sequence analysis of the RNA segment revealed that ts7 had four amino acid changes in the NA molecule but not in NB. We suggest that assembly or budding of influenza B virus requires the presence of NA at the plasma membrane, unlike influenza A virus.     

Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins.

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.     

Influenza virus assembly and its defects

The most abundant protein within the influenza virus particles is membrane protein (M protein) which forms an inner virus membrane under a lipid bilayer and plays the role of mediator during the process of assembly of a virus particle on plasma membranes. Ehrlich ascites tumor cells (EAT) when infected with influenza virus, strain WSN, produced virus-like particles containing greatly reduced amounts of M protein. Such particles were extremely fragile and easily lost hemagglutinins. The loss of this glycoprotein was accompanied by a decrease in infectious activity.SDS-PAGE analysis of RNA duplexes formed after hybridization of intracellular labeled mRNAs and unlabeled virion RNA showed that the mRNA for M protein was synthesized in EAT nearly in the same amounts as in productively infected chicken fibroblasts. Accordingly, M protein was readily revealed when the polypeptides of infected EAT were analyzed by SDS-PAGE. Thus, the reduced amount of M protein in virus particles was likely not due to the decrease in its synthesis but rather to its defective structure or to its defective transport and misintegration into plasma membranes of EAT.     

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.     

Molecular defects caused by temperature-sensitive mutations in Semliki Forest virus nsP1

Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.     

Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.     

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus.

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phase M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.     

携带TAP标签的重组甲型流感病毒的构建与鉴定

【目的】将TAP标签构建到WSN病毒基因组上,得到含有TAP标签的重组流感病毒,以便进行后续的病毒追踪。【方法】利用反向遗传学技术,对甲型流感病毒A/WSN/33(H1N1)的PA片段进行改造来插入TAP(tandemaffinitypurification)标签序列。通过病毒拯救得到表达外源标签TAP的重组流感病毒WSNPA-TAP,并对拯救出的重组病毒进行生物学鉴定。【结果】成功拯救出重组流感病毒并命名为WSN PA-TAP。重组病毒基因组测序表明重组病毒的序列正确,利用RNA银染技术观察到重组病毒的全基因组片段。重组流感病毒WSN PA-TAP在MDCK细胞上测定生长曲线,发现该重组病毒的复制能力比野生型WSN弱;Westernblotting检测到PA-TAP融合蛋白的表达,其分子质量为96 kDa。【结论】成功拯救出能够表达外源标签TAP的重组流感病毒WSN PA-TAP,为筛选与甲型流感病毒聚合酶有关的宿主蛋白的研究提供了新思路,同时也为以甲型流感病毒为载体携带外源基因的探索提供了重要依据。