Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides

An influenza A virus, A/turkey/Oregon/71, was shown by protein gel analysis to code for an NS1 protein approximately half the size of those of other influenza A viruses. Sequence analysis of the NS gene of this virus revealed a 10 nucleotide deletion resulting in an NS1 protein of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by indirect immunofluorescence analysis of virus infected cells. Also, A/turkey/Oregon/71 virus grew to high titer in embryonated chicken eggs comparable to other influenza A viruses. We also identified a laboratory variant of an influenza B virus, clone 201, which codes for a truncated NS1 protein. Sequence analysis revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids as compared to other influenza B virus NS1 proteins possessing a length of 281 amino acids. Again as shown for the NS1 proteins of other influenza B viruses the NS1 polypeptide of B virus clone 201 was found to localize in the nucleus of infected cells. It appears that large deletions in the carboxyl terminus of the NS1 proteins of influenza A and B viruses can be tolerated without affecting the functional integrity of the NS1 polypeptide.     

Intracellular localization of influenza C virus NS2 protein (NEP) in infected cells and its incorporation into virions

RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2. The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP). In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions. Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection. Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography. Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope. Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.     

Loss of function of the influenza A virus NS1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (PI3K)

A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.     

Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.     

The NS1 protein of H5N6 feline influenza virus inhibits feline beta interferon response by preventing NF-κB and IRF3 activation

Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-β promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-β suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.     

Rescue of influenza virus expressing GFP from the NS1 reading frame

In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA

Simian cells permissive for influenza A virus infection were stably transformed with a full-length cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate cell selection. Transformed cells synthesized a virus-specific nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to molecular weight and intracellular localization. It was estimated that transformed cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nonetheless, when transformed cells were infected with influenza virus mutants which synthesized temperature-sensitive nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for one mutant and only plus-strand synthesis for another mutant. This indicated that the influenza A nucleoprotein expressed in the transformed cells exhibited functional activity.     

Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells. Received March 7, 2000 Accepted August 16, 2000     

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.     

Multiple levels of PKR inhibition during HIV-1 replication

Recent therapeutic approaches against HIV-1 include IFN in combination therapy for patients with coinfections or as an alternative strategy against the virus. These treatment options require a better understanding of the weak efficacy of the IFN-stimulated genes, such as the protein kinase RNA-activated (PKR), which results in viral progression. Activated PKR has a strong antiviral activity on HIV-1 expression and production in cell culture. However, PKR is not activated upon HIV-1 infection when the virus reaches high levels of replication, due to viral and cellular controls. PKR is activated by low levels of the HIV-1 trans-activation response (TAR) RNA element, but is inhibited by high levels of this double-stranded RNA. The viral Tat protein also counteracts PKR activation by several mechanisms. In addition, HIV-1 replicates only in cells that have a high level of the TAR RNA binding protein (TRBP), a strong inhibitor of PKR activation. Furthermore, increased levels of adenosine deaminase acting on RNA (ADAR1) are observed when HIV-1 replicates at high levels and the protein binds to PKR and inhibits its activation. Finally, the PKR activator (PACT) also binds to PKR during HIV-1 replication with no subsequent kinase activation. The combination of all the inhibiting pathways that prevent PKR phosphorylation contributes to a high HIV-1 production in permissive cells. Enhancing PKR activation by counteracting its inhibitory partners could establish an increased innate immune antiviral pathway against HIV-1 and could enhance the efficacy of the IFN treatment.     

Inhibition of influenza C viruses by human MxA protein

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.     

The effect of the vaccinia K1 protein on the PKR-eIF2α pathway in RK13 and HeLa cells

Activated PKR protein regulates downstream anti-viral effects, including inhibition of translation. Thus, many viruses encode proteins to inhibit PKR. Here, we provide evidence that the vaccinia virus K1 protein, a host-range protein, possesses this function. First, the expression of the wild-type K1 protein was necessary to inhibit virus-induced eIF2α phosphorylation, an indirect measure of PKR activation, in RK13 and HeLa cells. Second, virus-induced eIF2α phosphorylation no longer occurred in PKR-deficient HeLa cells, suggesting PKR was responsible for vaccinia virus-induced eIF2α modification. Third, in normal HeLa cells, K1 protein expression also prevented virus-mediated PKR phosphorylation (activation). Residues in the C-terminal portion of the ANK2 region of K1 were identified as necessary for this inhibitory phenotype. Interestingly, mutant viruses that failed to inhibit PKR activation, such as S2C#2, also did not replicate in HeLa cells, suggesting that K1's inhibition of PKR was required for a productive infection. In support of this theory, when PKR was absent from HeLa cells, there was a modest restoration of viral protein synthesis during S2C#2 infection. However, the increased protein synthesis was insufficient for a productive infection.     

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.     

Characterization of a unique protein produced by influenza A virus recovered from a long-term persistent infection

Virus isolated from a persistent infection initiated in BHK cells with influenza A/WSN/33 (H1N1) produced an unusual pattern of protein synthesis in productive infections of BHK cells: The levels of NP and M1 proteins were slightly reduced compared to an infection with wild-type WSN, while the other proteins (Pb1, Pb2, Pa, HA, NS1, and NS2) were synthesized at very low or undetectable levels. In addition, a new viral protein with an approximate molecular weight of 11 kDa (Pi protein) is made (Frielle et al., Virology 138, 103-117, 1984). When viral RNA was analyzed by the Northern blot technique, a deletion was found in the NS gene segment and in NS1 mRNA; all other RNAs were full-sized. Immunoprecipitation of in vitro translation products demonstrated that the Pi protein reacts specifically with anti-NS1 serum. In addition, the Pi protein, like the NS1 of the parental wild-type virus, accumulated in the nucleus of infected cells. These results indicate that the Pi protein is a mutated form of the NS1 protein encoded by a deleted NS segment and suggest that this mutation may be involved in the expression of the persistent virus phenotype.