Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs.

The effect of NS1 protein on the efficiency of influenza virus mRNA translation was evaluated by determining the accumulation of nucleoprotein (NP) or M1 mRNAs in the cytoplasm of cells expressing either of these genes alone or in combination with the NS1 gene, as well as the total cell accumulation of NP or M1 protein. Coexpression of NS1, but not of NS2 protein, led to increases in the translation of these mRNAs in the range of 5- to 100-fold. This translation enhancement was specific for viral mRNAs, since the translation of neither cat nor lacZ mRNAs was affected by the coexpression of NS1 protein. The use of chimeric cat genes containing the 5'-extracistronic sequences of the influenza virus mRNAs corresponding to segment 2, 7, or 8 indicated that these sequences can in part account for the observed effect. The enhancement of viral mRNA translation mediated by NS1 protein was due to an increase in the translation initiation rate, since the sizes of NP-specific polysomes, but not those of lacZ-specific polysomes, was significantly higher in cells coexpressing NS1 protein than in those expressing only the NP gene.     

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.     

Attenuated influenza virus construct with enhanced hemagglutinin protein expression

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.     

The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A).

The influenza virus NS1 protein inhibits the nuclear export of a spliced viral mRNA, NS2 mRNA (F. V. Alonso-Caplen, M. E. Nemeroff, Y. Qiu, and R. M. Krug, Genes Dev. 6:255-267, 1992). To identify the sequence in NS2 mRNA that is recognized by the NS1 protein, we developed a gel shift assay for the formation of specific RNA-protein complexes. With this assay, it was established that the NS1 protein binds to the poly(A) sequence at the 3' end of NS2 mRNA and of other mRNAs. In addition, the NS1 protein was shown to bind to poly(A) itself. This specificity was also observed in vivo. The NS1 protein inhibited the nuclear export of every poly(A)-containing mRNA that was tested. In contrast, the NS1 protein failed to inhibit the nuclear export of an mRNA whose 3' end was generated by cleavage without subsequent addition of poly(A). Addition of poly(A) to this mRNA enabled the NS1 protein to inhibit mRNA export. The implications of these results for the role of the NS1 protein during virus infection are discussed.     

Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.     

The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells

The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.     

The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation.

A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.     

Selective nuclear export of viral mRNAs in influenza-virus-infected cells

The NS1A protein of influenza A virus specifically inhibits the cellular machinery that processes the 3' ends of cellular pre-mRNAs by targeting two of the essential proteins of this machinery. Because the virus does not use this cellular machinery to synthesize the 3' poly(A) ends of viral mRNA, the nuclear export of cellular but not viral mRNAs is selectively inhibited.     

Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein

The second gene in the 3′-to-5′ gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (ΔNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and ΔNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the ΔNS2 virus. Synthesis of readthrough mRNAs was affected only for the ΔNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant ΔNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the ΔNS2 RSV is a candidate for vaccine development.     

Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus

We have obtained the complete sequence of cloned full-length DNA (NS DNA) derived from influenza virus gene 8, which codes for two unique polypeptides, NS₁ and NS₂, and the sequence of the NS₂ mRNA. Previously we showed that the mRNA for NS₁ (～860 nucleotides) is colinear with the viral RNA and maps from 0.05?0.95 units of the cloned NS DNA, and the body of the NS₂ mRNA (～340 nucleotides) maps from 0.59?0.95 units, suggesting that the two mRNAs are 3′ co-terminal and share the same poly(A) addition site. Sequencing studies have shown that the NS₂ mRNA contains an interrupted sequence of 473 nucleotides. The nucleotide sequences at the junctions of the interrupted segment are similar to those of the consensus sequences at the splicing sites of intervening regions in eucaryotic mRNAs. The first ～56 virus-specific nucleotides at the 5′ end of the NS₂ mRNA are the same nucleotides as are found at the 5′ end of the NS₁ mRNA, and this leader sequence of the NS₂ mRNA contains the initiation codon for protein synthesis and coding information for nine amino acids which would be common to NS₁ and NS₂. In addition, both mRNAs contain 10–20 heterogeneous nonviral nucleotides at their 5′ ends. The ～340 nucleotide body region of the NS₂ mRNA can be translated in the +1 reading frame, and the sequence indicates that NS₁ and NS₂ overlap by 70 amino acids that are translated from different reading frames.     

Differential effects of NS1 proteins of human pandemic H1N1/2009, avian highly pathogenic H5N1, and low pathogenic H5N2 influenza A viruses on cellular pre-mRNA polyadenylation and mRNA translation

The nonstructural protein NS1 of influenza A virus blocks the development of host antiviral responses by inhibiting polyadenylation of cellular pre-mRNA. NS1 also promotes the synthesis of viral proteins by stimulating mRNA translation. Here, we show that recombinant NS1 proteins of human pandemic H1N1/2009, avian highly pathogenic H5N1, and low pathogenic H5N2 influenza strains differentially affected these two cellular processes: NS1 of the two avian strains, in contrast to NS1 of H1N1/2009, stimulated translation of reporter mRNA in cell-free translation system; NS1 of H5N1 was an effective inhibitor of cellular pre-mRNA polyadenylation in A549 cells, unlike NS1 of H5N2 and H1N1/2009. We identified key amino acids in NS1 that contribute to its activity in these two basic cellular processes. Thus, we identified strain-specific differences between influenza virus NS1 proteins in pre-mRNA polyadenylation and mRNA translation.     

Imaging and characterizing influenza A virus mRNA transport in living cells

The mechanisms of influenza A virus mRNA intracellular transport are still not clearly understood. Here, we visualized the distribution and transport of influenza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measurements determined that the transport of influenza A virus intronless mRNA, in both nucleus and cytoplasm, was energy dependent, being similar to that of Poly(A)⁺ RNA. Drug inhibition studies in living cells revealed that the export of influenza A virus mRNA is independent of the CRM1 pathway, while the function of RNA polymerase II (RNAP-II) may be needed. In addition, viral NS1 protein and cellular TAP protein were found associated with influenza A virus mRNA in the cell nucleus. These findings characterize influenza A virus mRNA transport in living cells and suggest that influenza A virus mRNA may be exported from the nucleus by the cellular TAP/p15 pathway with NS1 protein and RNAP-II participation.