Oriented synthesis and cloning of influenza virus nucleoprotein cDNA that leads to its expression in mammalian cells

The influenza virus nucleoprotein gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circularized by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA:dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known.     

Replication of animal viruses in differentiating muscle cells: influenza virus A

Cells were cultured from the breast muscle of 11- to 12-day-old chick embryos and were grown under conditions optimal for the development of the cells into terminally differentiated, fused myotubes. Myotubes were infected with influenza virus A/Ann Arbor/6/60(H2N2) at high multiplicity, and synthesis of virus-specific proteins and RNAs was detected by haemadsorption, fluorescence microscopy and/or isotope labelling and electrophoresis techniques. Provided that myotubes were maintained at temperatures below 39 degrees C after infection, production of virus components and yield of infectious virus in these cells was similar to those observed in infected chick kidney cells. However, if cells were maintained at temperatures of 39 degrees to 40 degrees C after infection, virus nucleoprotein was prominent in the nuclei, and synthesis of virus-specific polypeptides and of plus-strand RNA was reduced about fourfold to 20-fold compared to that detected at lower temperatures. Moreover, infectious virus was not produced when temperatures of 39 to 40 degrees C were used during virus replication. The results demonstrate that under suitable conditions avian myotubes formed in culture resemble epithelioid cells in their ability to support the productive replication of influenza virus.     

Cytoplasmic and nucleoplasmic viral RNPs in influenza virus-infected MDCK cells

In canine kidney (MDCK) cells infected with the WSN strain of influenza virus, viral ribonucleoproteins (RNPs) containing newly synthesized RNA and protein were first apparent in both the nucleoplasm and the cytoplasm at 2 hr. The viral RNPs in the nucleoplasm contained predominantly viral plus-strand RNA, whereas those in the cytoplasm consisted of two populations, one containing plus-strand RNA and the other containing minus-strand RNA. The nucleoplasmic viral RNPs containing plus-strand RNA were not the precursors for the cytoplasmic viral RNPs containing plus-strand RNA. At all times, only viral RNPs containing plus-strand RNA were incorporated into virus.     

Transcription and replication of eight RNA segments of influenza virus

A novel quantitation system of both plus- and minus-strand RNAs for all eight genome segments of influenza virus was developed using single-strand cDNAs as the probes for hybridization, and employed for the measurement of various RNA species in influenza virus WSN-infected MDBK cells. The synthesis rate and accumulation level of plus-strand RNAs differed considerably among eight RNA segments and were under temporal control. In contrast, eight vRNA molecules of minus polarity were synthesized coordinately at similar rates. Newly synthesized plus-strand RNAs were rapidly transported into the cytoplasm, particularly during the early phase of virus infection, but vRNAs accumulated in the nuclei until the late infection phase. The present data supported the differential regulation of synthesis and the separate transport between plus- and minus-strand RNAs.     

Recognition of out-of-frame major histocompatibility complex class I-restricted epitopes in vivo

In the course of constructing a recombinant vaccinia virus encoding the influenza A nucleoprotein (NP) gene preceeded by the hemagglutinin leader sequence, we isolated a single base-pair deletion mutant which gave rise to L+NP(1–159) in which only the first 159 amino acids were in frame. Despite this, when we infected target cells, we found that the point mutant was able to sensitize them for lysis not only by cytotoxic T cells recognizing residues 50–58 (the in-frame portion), but also by CTL to epitopes which are downstream of the mutation (366–374 and 378–386). Furthermore, normal C57BL/6 mice can be primed with the frameshift NP to recognize the immunodominant Db-restricted epitope 366–374 (which is out of frame). Experiments in which the mutant gene product was processed in the endoplasmic reticulum of target cells suggested that the apparent suppression occured during polypeptide extension.     

Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.     

Avian and human influenza a virus strains possess different intracellular nucleoprotein oligomerization efficiency

We have previously shown (Prokudina-Kantorovich EN and Semenova NP, Virology 223, 51-56, 1996) that the nucleoprotein (NP) of influenza A virus forms in infected cells oligomers which in the presence of SDS and 2-mercaptoethanol (ME) as reducing agent are stable at room temperature (RT) and dissociate at 100 degrees C. Here we report that the efficiency of intracellular NP oligomerization depends on the host origin of influenza A virus strain. Thus, in the cells infected with avian influenza A virus strains the viral NP was almost completely oligomerized and only traces of monomeric NP were detected by polyacrylamide gel electrophoresis (PAGE) in unboiled samples. However, in the cells infected with human influenza A virus strains, besides oligomeric NP also a significant amount of non-oligomerized monomeric NP was detected in unboiled samples. In purified virions of avian and human strains the same difference in NP monomers/oligomers ratio was detected as in the infected cells. A reassortant having all internal protein genes from a human strain and the glycoprotein genes from an avian strain revealed the same intracellular pattern of NP monomers/oligomers ratio as its parental human virus. These findings suggest that the type of NP oligomerization is controlled by the NP gene. The possible connection between the accumulation of protease-sensitive monomeric NP in cells infected with a human influenza strain and the parallel accumulation of cleaved NP in these cells is discussed.     

Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.     

Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live-attenuated vaccine candidate in the mouse model

A temperature-sensitive mutant virus unable to replicate at 38 degrees C was recovered from passage 189 (IVpi-189) of Madin-Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi-189 virus-infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non-permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61-24-P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi-189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi-189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi-189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi-189 virus, an avirulent temperature-sensitive mutant, is a promising candidate for use as a live-attenuated vaccine.     

Influenza B virus genome: complete nucleotide sequence of the influenza B/lee/40 virus genome RNA segment 5 encoding the nucleoprotein and comparison with the B/Singapore/222/79 nucleoprotein

The complete nucleotide sequence of a cloned full-length DNA copy of genome RNA segment 5 of influenza B/Lee/40 virus has been determined. The genome segment is 1841 nucleotides in length and is capable of coding for a nucleoprotein (NP) of 560 amino acids. Comparison with the only other known sequence of an influenza B virus nucleoprotein gene (B/Singapore/222/79) indicates striking homology. Only 113 nucleotide substitutions are present between the two strains in their protein coding region and these lead to only 22 amino acid substitutions between nucleoproteins of identical polypeptide chain length. Assuming a common lineage, this reflects a calculated rate of amino acid sequence divergence of 0.1% per year. Like its influenza A virus counterpart, the influenza B/Lee/40 nucleoprotein is a basic protein with a relatively even distribution of its charged residues. The remarkable conservation of nucleoprotein primary structure over a 39-year period probably reflects both selection for performance of specific functions and protection from antigenic selection by the host immune system.     

The influenza c virus glycoprotein (HE) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities

A cDNA copy of RNA segment 4 of influenza C/Cal/78 virus was cloned into an SV40 vector and expressed in CV-1 cells. The gene product expressed from the SV40 recombinant virus was immunoprecipitated by monoclonal antibodies directed against the influenza C virus glycoprotein. Cells infected with the recombinant virus also exhibited C virus-specific hemagglutinin and O-acetylesterase activity. This suggests that the same C virus protein is associated with receptor-binding as well as receptor-destroying activity. The latter viral activity was measured using as substrates bovine submaxillary mucin or a low molecular weight compound p-nitrophenylacetate. In analogy to the parainfluenza virus HN protein, the influenza C virus glycoprotein was termed HE, because it possesses hemagglutinin and esterase (receptor-destroying) activity.     

Complete nucleotide sequence of the tick-borne, orthomyxo-like Dhori/Indian/1313/61 virus nucleoprotein gene

The complete nucleotide sequence of the fifth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Ind/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. The fifth RNA contains 1479 nucleotides and can code for a protein of 477 amino acids with a molecular weight of 53,679 Da. The RNA 5 protein of the Dhori/Ind/1313/61 virus possesses five short regions (16-26 amino acids) which share a high degree (50-59%) of amino acid sequence homology with a computer-aligned consensus sequence of the influenza nucleoprotein gene family. These and other structural features of the RNA 5 protein suggest that RNA 5 of Dhori viruses codes for the nucleoprotein. The data also suggest that Dhori viruses are orthomyxoviruses, but that they are more distantly related to the influenza viruses than type A, B, and C viruses are to each other.     

Assessment of the specificity of cytotoxic T lymphocytes for the nucleoprotein of Pichinde virus using recombinant vaccinia viruses

Summary Pichinde virus (PV) infection of mice results in induction of a strong H-2 restricted, virus-specific cytotoxic T lymphocyte (CTL) response and rapid clearance of the virus. To define the specificities of CTL induced by PV infection, we constructed vaccinia virus recombinants containing cloned cDNAs corresponding to full-length (VVNP) and a truncated form (VVNP_51–561) of the nucleoprotein (NP) gene of PV. Radioimmunoprecipitation analysis of infected cell lysates indicated that VVNP expressed a PV-specific product identical in size to that of authentic NP, while vaccinia virus recombinants containing truncated NP produced a polypeptide consistent with the synthesis of amino acids 51–561 of Pichinde virus NP. Interestingly, cells infected with VVNP synthesized easily detectable, but much lower levels of nucleoprotein relative to both PV and VVNP_51–561. Primary virus-specific CTL induced in three different strains of inbred mice following intravenous infection with PV were able to lyse syngeneic target cells infected with PV but did not markedly lyse syngeneic targets expressing full-length or truncated NP following recombinant vaccinia virus infection. Similarly, secondary anti-PV specific CTL generated following in vitro restimulation by PV or selectively restimulated with vaccinia recombinants did not significantly lyse target cells expressing NP. Further, infection of mice with VVNP and VVNP_51–561 did not induce CTLs specific for PV and did not prime mice for the generation of memory anti-PV CTL in vivo. These results suggest that PV gene products other than NP, such as the GPC or L protein, contain the major target epitope(s) recognized by PV-specific CTL.     

Vaccinia virus expression and sequence of an avian influenza nucleoprotein gene: potential use in diagnosis

Summary The nucleoprotein (NP) gene from avian influenza strain A/Shearwater/Aust/1/72 (H6N5) was cloned, sequenced, and expressed in vaccinia virus for the production of potent sera in immunised rabbits. The NP gene is 1565 bp and shares >95% amino acid sequence identity with other NPs of the avian subtype. The recombinant NP expressed by vaccinia virus comigrated with endogenous A/Shearwater/Aust/1/72 NP by Western blot analysis. Polyclonal rabbit sera raised against recombinant NP was evaluated in an antigen capture ELISA system as a potential diagnostic tool for the detection of avian influenza. All type A strains, comprising several HA and NA subtypes, but not type B nor other avian viruses, were detected.     

以新型杆状病毒载体在昆虫、哺乳动物细胞中表达克里米亚-刚果出血热病毒核蛋白基因

目的 构建可以在哺乳动物细胞中高效表达的新型杆状病毒载体，并利用其将克里米亚—刚果出血热病毒(CCHFY)中国分离株(新疆出血热病毒，xHFY)BA88166的核蛋白(NP)基因在昆虫和哺乳动物细胞中进行表达。方法 将人巨细胞病毒(CMv)立即早期(IE)启动子连接至杆状病毒载体PFastBacl多角体启动子下游形成新载体PCB1，然后将xHFY NP基因克隆至该载体，通过重组质粒转染和病毒感染，检测其在哺乳动物细胞(COS—7和vero)及昆虫细胞中的表达。结果 连接至PCB1的xHFV NP基因均能在相应的细胞中获得良好表达；以重组杆状病毒感染的vero细胞可以作为抗原检测xHF血清，与ELISA的检测结果完全一致，并与临床诊断有很好的平行性。结论 新型杆状病毒载体能够驱动外源基因在昆虫和哺乳动物细胞中高效表达，不仅能方便快速地制备诊断抗原，还具有发展重组病毒疫苗和基因治疗的潜力。     

Influenza type A virus M protein expression on infected cells is responsible for cross-reactive recognition by cytotoxic thymus-derived lymphocytes.

M protein of influenza A virus was detected with rabbit antiserum by both indirect immunofluorescence and by antibody plus complement-mediated cytolysis on the cell surfaces of both productively and nonproductively infected cells. In contrast, antiserum to nucleoprotein failed to react with unfixed infected cells, but did bind to fixed infected cells, especially in the perinuclear area. Incorporation of antiserum to M protein in a T-cell-mediated cytotoxicity assay produced almost complete abrogation of lysis of H-2-compatible cells infected with an influenza A virus of a subtype which differed from that used to elicit the cytotoxic T cells. However, the antibody did not significantly block 51Cr release from cells infected with the homotypic type A influenza virus. These observations are in accord with the hypothesis that the cross-reactive cytotoxic T-cell responses seen with cells infected by heterotypic influenza A viruses are due to recognition of a common M protein.     

In vitro growth profiles of respiratory syncytial virus in the presence of influenza virus

To elucidate epidemiological interference between respiratory syncytial (RSV) and influenza viruses, the influence of influenza A (HlN1) virus on the growth of RSV was examined. Although RSV grew in MDCK cells, coinfection with influenza A virus led to a reduction of progeny RSV. The degree of growth interference depended on the time of infection with influenza A virus post infection (p.i.) with RSV. In fact, infection with influenza A virus 12 hrs p.i. with RSV did not influence growth of the latter virus. On the contrary, growth suppression of influenza A virus by RSV was observed when the coinfection began at the later stages of RSV infection. Suppression of the growth of RSV by influenza A infection was further demonstrated at the level of viral protein synthesis. An indirect immunofluorescence (IF) test revealed that a large proportion of infected cells synthesized both RSV and influenza A virus antigens. Scanning electron microscopic (SEM) examination demonstrated that influenza A and RSV virions possessing surface antigens specific for each virus were selectively released from dually infected cells. In the present study, we proved for the first time that the growth of RSV is blocked by competitive infection with influenza A virus in a susceptible cell population, competitive protein synthesis and selective budding of RSV and influenza viruses from the same infected cells.     

Inhibitory effect of diamidinophenylindole on the replication of influenza virus in permissive cellular hosts

Summary The production of avian and human strains of influenza virus was altered to various extents by treatment of various host cells with 40 µg/mI of diamidinophenylindole (DAPI). In infected LLC-MK 2 cells only an abortive replication cycle occurred; in other cell lines there was partial inhibition or no inhibition of replication. Virus polypeptide synthesis in LLC-MK 2 cells was confined to the early pattern of viral multiplication; only the P proteins, the nucleoprotein NP, and the non-structural protein NS 1 were synthesized. The stage of replication mainly affected by DAPI was between the fourth and the sixth hour after infection.The mode of action of the drug and its modulating effect on virus production is discussed.With 2 FiguresThe results reported here were in part presented at the sixth International Congress of Virology held in Senday, Japan, September 1–7, 1984.     

Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.     

Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus

The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.