**2.3. Replicase proteins**

the main limiting factor. Thus, viruses with combinations of polymerase factors that are most efficient will become dominant. One report of recombinants generated in eggs without selection described a preference for cosegregation of the HA and M segments [28]. Although the number of reports regarding reassortment without selection is limited, the current data suggests that egg-based experiments may more closely reflect naturally occurring events.

218 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

The replicase proteins may play a role in reassortment via their independent interaction with each RNA segment. Each genomic segment has it's own replicase proteins associated with it when it enters the nucleus. A doubly infected cell is capable of producing each of the segments from both viruses independently of each other. The timing of the overall replication will depend on many factors such as which segments are imported into the nucleus first, translation promoter sequences and replication signals on each segment, and the induced host response. If a cell is simultaneously infected with two viruses, it is possible that early in the infection the polymerase that transcribes RNA more efficiently will control the dynamics of the infection. The resulting dynamics between the host cell and the viruses may favor the production of either virus or a reassortant. It is unknown if the dynamics of transcription and replication play a role in reassortment but, given that the transciption and replication signals on each segment can differ, it is easy to imagine the dynamics of an infection being altered when a cell is infected with two viruses. One could also imagine polymerase proteins from one strain transcribing or replicating another strain's RNA, or a polymerase protein from one virus interacting with, and altering the activity of, a polymerase protein from the other virus.

There are two major surface glycoproteins for the influenza A and B viruses. The haemagglu‐ tinin (HA) protein is a sugar-binding protein that facilitates virus entry into epithelial cells that have sialic acid sugars on the cell surface. After the HA is cleaved by a protease, the virion is imported into the cell by endocytosis. Virus replication culminates with the accumulation of new virions at the cell surface. The neuraminidase (NA) cleaves the glycosidic linkages of the sialic acids to mediate virion release. The HA protein is encoded by segment 4 and the NA protein by segment 6 in influenza A and B viruses. Influenza C has only seven segments. The haemagglutinin-esterase-fusion (HEF) glycoprotein is encoded on segment 4 and this protein

In contrast to the influenza B and C viruses which are only sorted by type and strain, the influenza A viruses are sorted into subtypes. The HA and NA proteins are used for virus classification. There are at least 16 different HA subtypes and 9 different NA subtypes among the influenza A viruses. The HA subtypes are divided into two groups. Certain subtypes from both groups are able to infect and transmit among humans. Most human influenza A infections are from H1N1, H2N2, and H3N2 subtypes. Occasionally a strain will jump the species barrier. A limited number of avian subtypes (H5, H7 and H9) have infected humans. Sometimes the disease is much more severe than that from a human influenza strain but these strains seem to lack the ability to transmit from human to human efficiently. There is a fear that these highly virulent viruses may reassort with human viruses creating virulent viruses that spread easily among humans. New pandemic strains arising by reassortment is clearly a concern. An

performs the functions analogous to HA and NA of influenza A and B viruses.

**2.2. Glycoproteins**

Four proteins are required for influenza virus replication; the nucleocapsid protein (NP) and the three polymerase proteins PB1, PB2 and PA. The polymerase proteins are the larger influenza proteins and are encoded in the largest segments 1-3. NP is encoded in segment 5. The RNA from each viral segment form ribbon-like closed superhelical structures (reviewed in [30]). The 5' and 3' ends of the RNA are in close proximity to one another and are associated with the three polymerase proteins. Nucleoprotein is associated with the remaining genomic RNA and there is one NP monomer present for each 24 nucleotides. Nuclear localization sequences on NP facilitate import of each ribonucleoprotein complex (RNP) into the nucleus (reviewed in [31]). Inside the nucleus mRNA transcription and viral replication take place.

Two of the polymerase proteins, PB1 and PB2, have biochemical interactions with NP proteins. In addition, the three polymerase proteins interact with each other. The carboxyl-terminus of PA interacts with the amino-terminal end of PB1 and the carboxyl-terminal end of PB1 interacts with the amino-terminal end of PB2. The same arrangement is described for both the negative strand viral RNA (vRNP) and the positive strand copy RNA (cRNP) with the polymerase being associated with the 5' end of the RNAs. New negative-strand viral genomes are derived from the cRNP and also have a newly synthesized polymerase complex associated with the 5' end. The NP and three polymerase proteins all have nuclear localization signals which enable them to be imported into the nucleus after they are synthesized in the cytoplasm. There is evidence that PA and PB1 associate with each other before localizing to the nucleus (reviewed in [31]).

Both types of positive-strand RNA, cRNP and mRNA, are generated from the vRNP. In constrast to the vRNP and cRNP, the mRNA is not associated with NP. The viral mRNAs also have a capped 5' leader sequence snatched from a cellular mRNA and a polyadenylated 3' end. It is not yet known exactly what regulates the polymerase complex so that it makes two distinct products from one template. The cap-binding domain is in PB2 and the endonuclease domain is in PA. Together these parts of the polymerase complex capture and remove the 5' capped region of cellular mRNA and this is used as the priming sequence for viral mRNA production. The RNA-dependent RNA polymerase domain required for all RNA production is in the PB1 protein.

Combining the polymerase proteins from different strains to produce chimeric polymerase complexes has been studied with regard to polymerase activity and pathogenicity. It is sometimes found, but not always, that increased polymerase activity leads to more virus and increased pathogenicity [32]. Most recent studies have focused on the replicase genes from the 2009pandemic H1N1 strainandprior seasonal strains.It was foundthatthepandemicPB2 gene combinedwithseasonalPB1,PAandNPgenes resultedinsignificantlylesspolymerase activity [33;34]. Conversely, inclusion of a seasonal PB2 gene in a pandemic background significantly increased polymerase activity. When the corresponding reassortant viruses, with a PR/8 backbone, were generated the growth kinetics for both types were reduced. This suggests that the level of polymerase activity needs to be optimized for the best virus production in vitro. In addition these viruses had higher mouse LD50 values suggesting polymerase activity and replicationare importantforvirulence [33].Interestingly,introductionofthepandemicNPgene into a seasonal virus also dramatically reduced the virus replication and pathogenicity demonstrating that both altered polymerase and RNP could give rise to detrimental gene constellation effects.

tion of replication efficiency or associated pathogenicity based on replicase gene sequence alone. While new functional information is being generated regularly, a more complete understanding of influenza replication and its contribution toward pathogenicity will require

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There are at least four additional proteins produced during influenza infection of a cell. The two segments not mentioned so far, segments 7 and 8, are the smallest genome segments. In both influenza A and B viruses each of these segments encodes at least two proteins. The

Influenza A segment 7 encodes two matrix proteins; M1 and M2. The M2 proteins from influenza B and C viruses are called BM2 and CM2 respectively. M1 binding to RNPs in the nucleus inhibits viral transcription [42;43]. M1 proteins form a continuous shell on the inner side of the lipid bilayer. M2, and the analogous BM2 and CM2 proteins, are ion-channel proteins that form as a homotetramers in the virus envelope. These small hydrophobic integral membrane proteins allow hydrogen ions to enter the viral particle from the endosome. The lower pH causes M1 to dissociate from the RNPs leading to the uncoating of the virus. Different coding strategies are used by the different influenza species (Figure 2 and [44]). M2 protein is translated from a spliced transcript while BM2 protein is translated by a coupled termination/ reinitiation event [45;46]. In contrast CM1 is translated from a spliced transcript and CM2 is the produced by peptidase cleavage of a precursor protein [47;48]. Influenza B viruses encode an additional small hydrophobic integral membrane protein on segment 6. The open reading frame starts 4 nucleotides upstream from the NA ORF (Figure 2). Although NB is conserved in influenza B genomes it is apparently not essential and it's function remains unknown at present [49]. Interestingly, influenza A viruses also encode an alternative M2 protein on a splicing variant [3]. The same conserved redundancy in two different influenza families highlights the importance of the ion channels for the viruses. This diversity in coding and expression of similar functions may also be a reason why reassortant viruses containing

The smallest influenza segment encodes the non-structural protein NS1 and the nuclear export protein xlink. xlink is translated from a spliced transcript and is incorporated into the virions in small numbers (Figure 2). The major role of NS1 is to modulate the host immune response. It is a multifunctional protein that interacts with several host proteins and has an RNA binding domain (reviewed in [50]). Protein sequence features from influenza NS1 proteins indicate that there are variant types that seem to correlate with certain host species [51]. The exact nature of this relationship has not been teased out yet. The NS1 protein from the 2009 pandemic strain was less effective at blocking the innate immune response in cultured cells than other seasonal strains but attempts to make the 2009 NS1 protein more like the seasonal strain did not result in the same effect, rather the virus had reduced the virulence and was more easily cleared [52]. A better understanding the relationship between NS1 from specific virus strains and the host cell type could lead to the development of vaccine seed virus backbone strains that are more

analogous segments in influenza C viruses are segments 6 and 7 (Figure 1).

segments from different influenza types are not readily obtained.

suitable for vaccine production.

more comprehensive structure-function information.

**2.4. Other proteins**

In an analysis of ressortant viruses with a 2009 pandemic strain background it was found that introduction of a PA, PB1 or PB2 segment from another virus typically reduced the virus titer [27;35]. This included instances when all three segments from another virus increased polymerase activity (A/swine/Korea/JNS06/04 or A/mallard/Korea/6L/07) or reduced poly‐ merase activity (A/duck/Korea/LPM91/06 or A/aquatic bird/Korea/ma81/07). Again, this suggests that the level of polymerase activity needs to be optimized for efficient virus pro‐ duction in vitro. Each of these viruses were less pathogenic in mice but several other viruses were generated that were more pathogenic in mice. One, containing the just the PA segment from A/aquatic bird/Korea/ma81/07 in the 2009 pandemic backbone had a similar level of polymerase activity to the reassortant virus containing all three A/aquatic bird/Korea/ma81/07 polymerase genes indicating that polymerase activity per se is not the cause of pathogenicity [35]. It is possible that specific virulence determinants are associated with the PA segment but, in the absence of a gene constellation effect, this would not account for the lower pathogenicity of the virus containing all three polymerase segments.

One PB2 virulence marker, the amino acid at position 627, is a determinant of host range and contributes topathogenicity inmice [36].Ithas beenshownthatintroductionof aPB2 gene from a low pathogenic H1N1 virus into thehighlypathogenic 1918 strainattenuatedthe virus inmice but pathogenicity was restored with a E627K mutation [37]. In contrast, studies of swine influenza in pigs have shown that there is no correlation between pathogenicity and viruses with either a swine- or avian-origin PB2 gene containing the 627K or 627E mutation [38]. While it has been suggestedthatthe 627 residue mediates an interaction with NP[39], andthe strength of this interaction correlates with polymerase activity [40], recent evidence suggests that restricted activity is due to a lack of compatibility with a host cell factor [41]. Thus, although amino acid signatures of virulence may be importantin the context of genetic drift,these results demonstrate that gene constellation effects can attenuate virulence in some hosts.

Clearly, although specific functions of replicase complex reside in each protein, the interaction of the replicase proteins plays a large role in several virus attributes including replication and virulence. At present there is a lot of genetic information for the replicase genes available. Unfortunately our current understanding of influenza replication does not enable the predic‐ tion of replication efficiency or associated pathogenicity based on replicase gene sequence alone. While new functional information is being generated regularly, a more complete understanding of influenza replication and its contribution toward pathogenicity will require more comprehensive structure-function information.

## **2.4. Other proteins**

sometimes found, but not always, that increased polymerase activity leads to more virus and increased pathogenicity [32]. Most recent studies have focused on the replicase genes from the 2009pandemic H1N1 strainandprior seasonal strains.It was foundthatthepandemicPB2 gene combinedwithseasonalPB1,PAandNPgenes resultedinsignificantlylesspolymerase activity [33;34]. Conversely, inclusion of a seasonal PB2 gene in a pandemic background significantly increased polymerase activity. When the corresponding reassortant viruses, with a PR/8 backbone, were generated the growth kinetics for both types were reduced. This suggests that the level of polymerase activity needs to be optimized for the best virus production in vitro. In addition these viruses had higher mouse LD50 values suggesting polymerase activity and replicationare importantforvirulence [33].Interestingly,introductionofthepandemicNPgene into a seasonal virus also dramatically reduced the virus replication and pathogenicity demonstrating that both altered polymerase and RNP could give rise to detrimental gene

220 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

In an analysis of ressortant viruses with a 2009 pandemic strain background it was found that introduction of a PA, PB1 or PB2 segment from another virus typically reduced the virus titer [27;35]. This included instances when all three segments from another virus increased polymerase activity (A/swine/Korea/JNS06/04 or A/mallard/Korea/6L/07) or reduced poly‐ merase activity (A/duck/Korea/LPM91/06 or A/aquatic bird/Korea/ma81/07). Again, this suggests that the level of polymerase activity needs to be optimized for efficient virus pro‐ duction in vitro. Each of these viruses were less pathogenic in mice but several other viruses were generated that were more pathogenic in mice. One, containing the just the PA segment from A/aquatic bird/Korea/ma81/07 in the 2009 pandemic backbone had a similar level of polymerase activity to the reassortant virus containing all three A/aquatic bird/Korea/ma81/07 polymerase genes indicating that polymerase activity per se is not the cause of pathogenicity [35]. It is possible that specific virulence determinants are associated with the PA segment but, in the absence of a gene constellation effect, this would not account for the lower pathogenicity

One PB2 virulence marker, the amino acid at position 627, is a determinant of host range and contributes topathogenicity inmice [36].Ithas beenshownthatintroductionof aPB2 gene from a low pathogenic H1N1 virus into thehighlypathogenic 1918 strainattenuatedthe virus inmice but pathogenicity was restored with a E627K mutation [37]. In contrast, studies of swine influenza in pigs have shown that there is no correlation between pathogenicity and viruses with either a swine- or avian-origin PB2 gene containing the 627K or 627E mutation [38]. While it has been suggestedthatthe 627 residue mediates an interaction with NP[39], andthe strength of this interaction correlates with polymerase activity [40], recent evidence suggests that restricted activity is due to a lack of compatibility with a host cell factor [41]. Thus, although amino acid signatures of virulence may be importantin the context of genetic drift,these results

Clearly, although specific functions of replicase complex reside in each protein, the interaction of the replicase proteins plays a large role in several virus attributes including replication and virulence. At present there is a lot of genetic information for the replicase genes available. Unfortunately our current understanding of influenza replication does not enable the predic‐

demonstrate that gene constellation effects can attenuate virulence in some hosts.

constellation effects.

of the virus containing all three polymerase segments.

There are at least four additional proteins produced during influenza infection of a cell. The two segments not mentioned so far, segments 7 and 8, are the smallest genome segments. In both influenza A and B viruses each of these segments encodes at least two proteins. The analogous segments in influenza C viruses are segments 6 and 7 (Figure 1).

Influenza A segment 7 encodes two matrix proteins; M1 and M2. The M2 proteins from influenza B and C viruses are called BM2 and CM2 respectively. M1 binding to RNPs in the nucleus inhibits viral transcription [42;43]. M1 proteins form a continuous shell on the inner side of the lipid bilayer. M2, and the analogous BM2 and CM2 proteins, are ion-channel proteins that form as a homotetramers in the virus envelope. These small hydrophobic integral membrane proteins allow hydrogen ions to enter the viral particle from the endosome. The lower pH causes M1 to dissociate from the RNPs leading to the uncoating of the virus. Different coding strategies are used by the different influenza species (Figure 2 and [44]). M2 protein is translated from a spliced transcript while BM2 protein is translated by a coupled termination/ reinitiation event [45;46]. In contrast CM1 is translated from a spliced transcript and CM2 is the produced by peptidase cleavage of a precursor protein [47;48]. Influenza B viruses encode an additional small hydrophobic integral membrane protein on segment 6. The open reading frame starts 4 nucleotides upstream from the NA ORF (Figure 2). Although NB is conserved in influenza B genomes it is apparently not essential and it's function remains unknown at present [49]. Interestingly, influenza A viruses also encode an alternative M2 protein on a splicing variant [3]. The same conserved redundancy in two different influenza families highlights the importance of the ion channels for the viruses. This diversity in coding and expression of similar functions may also be a reason why reassortant viruses containing segments from different influenza types are not readily obtained.

The smallest influenza segment encodes the non-structural protein NS1 and the nuclear export protein xlink. xlink is translated from a spliced transcript and is incorporated into the virions in small numbers (Figure 2). The major role of NS1 is to modulate the host immune response. It is a multifunctional protein that interacts with several host proteins and has an RNA binding domain (reviewed in [50]). Protein sequence features from influenza NS1 proteins indicate that there are variant types that seem to correlate with certain host species [51]. The exact nature of this relationship has not been teased out yet. The NS1 protein from the 2009 pandemic strain was less effective at blocking the innate immune response in cultured cells than other seasonal strains but attempts to make the 2009 NS1 protein more like the seasonal strain did not result in the same effect, rather the virus had reduced the virulence and was more easily cleared [52]. A better understanding the relationship between NS1 from specific virus strains and the host cell type could lead to the development of vaccine seed virus backbone strains that are more suitable for vaccine production.

The presence of segment 7 or 8 from differing viruses can alter the phenotype of another virus. For example, addition of different NS segments from an H3N2 virus or different H5N1 viruses into a PR/8 backbone could result in no attenuation or complete attenuation [53;54]. Similarly, the same gene can have different effects on different viruses. For example, replacement of the A/Korea/82 (H3/N2) M segment with the A/Ann Arbor/6/60 M segment attenuated the virus. However, introducing the same A/Ann Arbor/6/60 M segment into A/Udorn/72 (H3/N2) did not attenuate the virus [55]. This clearly demonstrates the greater impact the gene constellation has toward the virus phenotype than an individual segment in this instance.

and colleagues to analyze the reassortants present in swine populations. What they discovered was that, in addition to the reassortants that transmitted to humans, reassortants in swine with a range of genetic backbones contained the 2009 pandemic segment 7 [61]. It is not known if the presence of the 2009 segment 7 in swine viruses plays a role in viral fitness in swine or if it has a role in zoonotic infection of humans, but clearly the presence of this segment gives rise to viruses from different reassortment events that are stably represented in the population.

In addition to the appearance of this particular gene constellation in North America pigs, gene constellations involving all segments from the 2009 pandemic strain are becoming dominant in other parts of the world. It has been reported that the 2009 pandemic strain is present in pigs and reassorting with H1N2 and H3N2 strains [62-65]. It is not clear how the combination of gene segments present in strains like the 2009 pandemic strain results in a greater viral fitness but study of different viral characteristics has given us some insight. Here we highlight

54 aa

99 aa

**Figure 2.** Coding strategies of influenza viruses (adapted from Lamb & Takeda, 2001). A) Multiple splice variants are transcribed from one segment to enable the production of multiple proteins. As many as four mRNAs (and proteins) are produced from influenza A segment 7. The mRNA from the smallest segment of all influenza species is spliced for the translation of the nuclear export protein xlink. B) A larger protein is translated and processed to produce the influ‐ enza C protein CM2. The M1 protein is produced from a spliced mRNA. C) Different AUG start codons are used to pro‐ duce up to three proteins from influenza A segment 1. A second membrane protein, NB, is produced from an alternative start codon in segment 6 of influenza B. D) The termination and start codons of the respective M1 and BM2 open reading frames in segment 7 of influenza B overlap. A stop/start translation strategy is used to produce the

A(n)

A(n)

NA 466 aa

A(n)

248 aa 109 aa

Polycistronic mRNAs: **A**/PB1/PB1-F2; **B**/NB/NA

PB1-N40

PB1

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Tandem cistrons –stop/start translation:

BM2

…UAAUG…

AUG

PB1-F2

AUG

C

AUG

AUG

AUG

**B**/M1/M2

AUG

D

NB

M1

AUG

100 aa

758 aa 718 aa 87 aa

research describing the effect different gene constellations have on viral fitness.

A(n) 347 aa

A(n) 242 aa

RNA 1 M1m A(n) 252 aa mRNA 2 M2 A(n) 97 aa

**A**/M1/M2/M42; **A/B/C**/NS1/NS2(NEP)

M3? mRNA 3 A(n) 9 aa mRNA 4 M42 A(n)

mRNA 1 NS A(n) 237 aa mRNA 2 NS2 A(n) 121 aa

Splicing and post-translational processing:

p31 CM2 p42

M1

M4?

Spliced mRNAs:

mRNA M1

A

mRNA 1

B

**C**/CM1/CM2

mRNA 2

second protein.

The characterization of the laboratory generated reassortants provides useful hypothesisdriven information. Natural reassortment of the smallest influenza segments may give additional information about the role these segments play in the virus lifecycle. Some viruses isolated recently from North American pigs contain the 2009 H1N1 M segment in the context of a previously endemic H1N2 strain [56]. This suggests that this particular gene constellation may increase viral fitness. Supporting this Chou et al., [57] were able to show that inclusion of the M segment in a PR/8 backbone was essential for transmission in guinea pigs. Another group reported that the neuraminidase segment from the 2009 H1N1 strain, in addition to the M segment, was required for efficient replication and transmission in pigs [58]. Finally, Hause et al. [59] reported transmission but lower viral titers for the reassortant viruses containing the M segment from the 2009 pandemic strain in pig lung homogenate when compared to infection with the parental strains. The major difference between the viruses analyzed by these groups is that the backbone strains differed; one group used comtemporary H1N2 swine viruses to generate reassortants while the other groups used laboratory adapted strains. The different outcomes observed are based on gene constellation effects.
