Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro.

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Early results suggested that the RNA polymerase "switched" from a transcriptase to a replicase during the viral life cycle in response to the expression of viral proteins. However, recently an alternative model suggesting that replication of influenza virus is regulated by stabilization of the replicative intermediates was proposed. According to this model, the virion-associated polymerase is capable of synthesizing both mRNA and cRNA. We now demonstrate that virion-derived viral ribonucleoproteins (vvRNPs) synthesize both mRNA and cRNA in vitro in the absence of non-virion-associated RNA polymerase or nucleoproteins. The authenticity of the in vitro-transcribed mRNA and cRNA was confirmed by terminal sequence analysis. The addition of non-virion-associated polymerase or NP had no effect on vvRNP activity. De novo synthesis of cRNA was found to be more sensitive than the capped primer-dependent synthesis of mRNA to the concentration of ATP, CTP, and GTP. We conclude that vvRNPs intrinsically possess both transcriptase and replicase activities and that there is no switch in the synthesis of mRNA to cRNA during the influenza virus life cycle.

Orthomyxovirus replication, transcription, and polyadenylation.

Efficient in vitro and in vivo systems are now in place to study the role of viral proteins in replication and/or transcription, the regulation of these processes, polyadenylation of viral mRNAs, the viral promoter structures, or the significance of noncoding regions for virus replication. In this chapter, we review the status of current knowledge of the orthomyxovirus RNA synthesis.

Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene.

The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.

Hairpin loop structure in the 3' arm of the influenza A virus virion RNA promoter is required for endonuclease activity.

Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.

DNA vaccination against respiratory influenza virus infection.

DNA vaccination using plasmid encoding the hemagglutinin (HA) gene of influenza A/PR/8/34 virus to induce long-lasting protective immunity against respiratory infection was evaluated in this study. Using liposomes as carriers, the efficacy of DNA vaccines was determined using a lethal influenza infection model in mice. Mice immunized intranasally or intramuscularly with liposome-encapsulated pCI plasmid encoding HA (pCI-HA10) were completely protected against an intranasal 5 LD(50) influenza virus challenge. Mice immunized with liposome-encapsulated pCI-HA10, but not naked pCI-HA10, by intranasal administration were found to produce high titers of serum IgA. These results suggest DNA vaccines encapsulated in liposomes are efficacious in inducing complete protective immunity against respiratory influenza virus infection.

Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity.

Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5' arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5' hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5' terminus, and (iii) the presence of base pair interactions between the 5' and 3' segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.

Reduced levels of neuraminidase of influenza A viruses correlate with attenuated phenotypes in mice.

We have previously obtained four transfectant influenza A viruses containing neuraminidase (NA) genes with mutated base pairs in the conserved double-stranded RNA region of the viral promoter by using a ribonucleoprotein transfection system. Two mutant viruses (D2 and D1/2) which share a C-G-->A-U mutation at positions 11 and 12 of the 3' and 5' ends, respectively, of the NA gene, showed an approximate 10-fold reduction of NA-specific mRNA and protein levels (Fodor et al., Journal of Virology 72, 6283-6290, 1998). These viruses have now allowed us to determine the effects of decreased NA levels on virus pathogenicity. Both D2 and D1/2 viruses were highly attenuated in mice, and their replication in mouse lungs was highly compromised as compared with wild-type influenza A/WSN/33 virus. The results highlight the importance of the level of NA activity in the biological cycle and virulence of influenza viruses. Importantly, mice immunized by a single intranasal administration of 10(3) infectious units of D2 or D1/2 viruses were protected against challenge with a lethal dose of wild-type influenza virus. Attenuation of influenza viruses by mutations resulting in the decreased expression of a viral protein represents a novel strategy which could be considered for the generation of live attenuated influenza virus vaccines.

Messenger RNAs that are not synthesized by RNA polymerase II can be 3' end cleaved and polyadenylated.

The poly(A) tail of influenza virus mRNAs is synthesized by the viral RNA polymerase by reiterative copying of a U5-7 sequence near the 5' end of the viral RNA (vRNA) template. We have engineered a vRNA molecule by replacing its viral U6 poly(A) site with a negative-sense eukaryotic polyadenylation signal. The vRNA was transcribed by the viral RNA polymerase and the transcription product was processed by the cellular 3' end processing machinery in vivo. According to the current model, 3' end processing of eukaryotic pre-mRNAs is coupled to cellular RNA polymerase II (pol II) transcription; thus only RNAs synthesized by pol III are believed to be polyadenylated efficiently. Our results show that the cellular polyadenylation machinery is nevertheless able to recognize and process RNA transcripts that are not synthesized by pol II, indicating that synthesis by pol II is not an absolute requirement for 3' end processing in vivo.

Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export.

The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5' end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.

Rescue of influenza A virus from recombinant DNA.

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.

Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template.

The poly(A) tail of influenza virus mRNA is thought to be synthesized by reiterative copying of the U track near the 5' end of the virion RNA template. This has been widely accepted as a plausible hypothesis, but until now there has been no direct experimental evidence for it. Here, we report such direct evidence based on the fact that (i) replacing the U track with an A track directs synthesis of products with poly(U) tails, both in vitro and in vivo, and (ii) interrupting the U track abolishes polyadenylation in vitro.

A hairpin loop at the 5' end of influenza A virus virion RNA is required for synthesis of poly(A)+ mRNA in vitro.

We present evidence, based on extensive mutagenesis, that a hairpin loop at the 5' end of influenza A virus virion RNA (vRNA) is required for the synthesis of polyadenylated mRNA from model vRNA templates in vitro. The hairpin loop, which we term the vRNA 5' hook, contains a stem of 2 bp formed by the second and third residues pairing with the ninth and eighth residues, respectively, and a 4-nucleotide loop composed of the intervening residues 4 to 7. Disruption of the base pairs of the vRNA 5' hook by introducing point mutations prevented polyadenylation, except in two mutants where a G-U base pair reformed. The polyadenylation activity of point mutants could be rescued by constructing double mutants with reformed base pairs in the stem of the vRNA 5' hook. These results suggest that base pairing rather than a particular nucleotide sequence was critical. We also show that mutation of the analogous region in the 3' arm of vRNA did not interfere with the synthesis of polyadenylated mRNA, suggesting that a hook structure in the 3' arm is not required for transcription of polyadenylated mRNA in vitro.

The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to polyadenylate mRNA.

We previously demonstrated, by limited mutagenesis, that conserved sequence elements within the 5' end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further characterize the nucleotide residues at the 5' end of vRNA which might be involved in polyadenylation, a complete set of short and long model vRNA-like templates with mutations at nucleotides 1' to 13' (prime notation denotes numbering from the 5' end) of vRNA were synthesized and transcribed in vitro. The products were assayed for mRNA production with both reverse transcription-PCR and [alpha-32P]ATP incorporation assays. Results from these independent assays showed that vRNA templates with point mutations at positions 2', 3', 7' to 9', and 11' to 13' synthesized polyadenylated transcripts inefficiently compared with those with mutations at positions 1', 4' to 6', and 10'. Positions 2', 3', 7' to 9', and 11' are known to be involved in RNA polymerase binding. Furthermore, residues at positions 11' to 13' are known to be involved in base pairing between the 3' and 5' ends of vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5' end of the template vRNA, which must then interact with the 3' end of the same template for polyadenylation to occur. These results support a model in which a cis-acting RNA polymerase is required for the polyadenylation of influenza virus.

Attenuation of influenza A virus mRNA levels by promoter mutations.

We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G-->A-U double mutation at positions 11 and 12' at the 3' and 5' ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

The activation function 2 domain of hepatic nuclear factor 4 is regulated by a short C-terminal proline-rich repressor domain.

Hepatic nuclear factor 4 (HNF4) is a transcription factor whose expression is crucial for mouse embryonic development, for liver-specific gene expression and for the prevention of one form of maturity-onset diabetes of the young. Its domain structure has been defined previously and is similar to other members of the nuclear receptor superfamily. A repressor domain has now been localised to a region of 14 amino acids (residues 428-441) near the C-terminus of HNF4 and is sufficient by itself to repress the activity of the activation function 2 (AF2) domain. Multiple mutations within this repressor domain enhance activity. Interestingly, this repressor domain shares homology with a repressor domain in the progesterone receptor. In a detailed mutagenesis study of the AF2 core, we demonstrate that L 366, which is conserved in the AF2 core between HNF4 and a number of orphan nuclear receptors, is essential for the full activity of the AF2 domain. Furthermore, a double mutation of E 363 and L 366 suggests that these residues might act in a cooperative manner.

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy.

HaCaT cells, a spontaneously immortalised, nontumorigenic keratinocyte line, were used as a more amenable model than primary keratinocytes for ex vivo-mediated gene transfer. These cells were transduced with retroviral vectors containing the factor IX cDNA under the control of a cytomegaloviral (CMV) promoter/enhancer alone or as hybrids with either the human papilloma virus-16 (HPV-16), keratin 14 (hK14) or keratin 5 (hK5) regulatory elements. Unlike primary keratinocytes, HaCaT cells tolerated transduction and G418 selection well. The HPV-16 and hK5 hybrid constructs were disproportionately more active in primary keratinocytes than in the basal-like HaCaT cells. After skin grafting to athymic mice, transduced HaCaT cells differentiated to form a stratified epidermis that remained viable for at least 99 days in some mice. Factor IX in plasma of mice grafted with vectors containing the HPV-16 and hK5 elements was two- to three-fold higher than with vectors containing the CMV promoter alone. These results are consistent with the expected up-regulation in differentiated suprabasal cells by the HPV-16 and hK5 elements. Enhancers may be useful in specifically targeting the differentiated layer of the epidermis or achieving higher levels of gene expression after transplantation.

Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.

In Xenopus oocytes, progesterone stimulates the cytoplasmic polyadenylation and resulting translational activation of c-mos mRNA, which is necessary for the induction of oocyte maturation. Although details of the biochemistry of polyadenylation are beginning to emerge, the mechanism by which 3' poly(A) addition stimulates translation initiation is enigmatic. A previous report showed that polyadenylation induced cap-specific 2'-O-methylation, and suggested that this 5' end modification was important for translational activation. Here, we demonstrate that injected c-mos RNA undergoes polyadenylation and cap ribose methylation. Inhibition of this methylation by S-isobutylthioadenosine (SIBA), a methyltransferase inhibitor, has little effect on progesterone-induced c-mos mRNA polyadenylation or general protein synthesis, but prevents the synthesis of Mos protein as well as oocyte maturation. Maturation can be rescued, however, by the injection of factors that act downstream of Mos, such as cyclin A and B mRNAs. Most importantly, we show that the translational efficiency of injected mRNAs containing cap-specific 2'-O-methylation (cap I) is significantly enhanced compared to RNAs that do not contain the methylated ribose (cap 0). These results suggest that cap ribose methylation of c-mos mRNA is important for translational recruitment and for the progression of oocytes through meiosis.

Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo.

A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/10(6) cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation.

Haemophilia B: database of point mutations and short additions and deletions--eighth edition.

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.