Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.Influenza A viruses contain eight negative-stranded RNA segments that encode 11 known viral proteins. The 11th viral protein was originally found in a search for unknown peptides during influenza A virus infection recognized by CD8⁺ T cells. It was termed PB1-F2 and is the second protein that is alternatively translated by the same PB1 gene (8). PB1-F2 can be encoded in a large number of influenza A viruses that are isolated from various hosts, including human and avian hosts. The size of PB1-F2 ranges from 57 to 101 amino acids (aa) (41). While strain PR8 (H1N1) contains a PB1-F2 with a length of 87 aa, PB1-F2 is terminated at amino acid position 57 in most human H1N1 viruses and is thus a truncated form compared with the length in PR8. Human H3N2 and most avian influenza A viruses encode a full-length PB1-F2 protein, which is at least 87 aa (7). Many cellular functions of the PB1-F2 protein, and especially the protein of the PR8 strain, have been reported (11, 25). For example, PR8 PB1-F2 localizes to mitochondria in infected and transfected cells (8, 15, 38, 39), suggesting that PB1-F2 enhances influenza A virus-mediated apoptosis in human monocytes (8). The phosphorylation of the PR8 PB1-F2 protein has been suggested to be one of the crucial causes of the promotion of apoptosis (30).The rates of synonymous and nonsynonymous substitutions in the PB1-F2 gene are higher than those in the PB1 gene (7, 20, 21, 37, 42). Recent work has shown that both PR8 PB1-F2 and H5N1 PB1-F2 are important regulators of influenza A virus virulence (1). Additionally, the expression of the 1918 influenza A virus (H1N1) PB1-F2 increases the incidence of secondary bacterial pneumonia (10, 28). However, PB1-F2 is not essential for viral replication because the knockout of PB1-F2 in strain PR8 has no effect on the viral titer (40), suggesting that PB1-F2 may have cellular functions other than those that were originally thought (29).PB1-F2 was translated from the same RNA segment as the PB1 protein, whose function is strongly related to virus RNP activity, which is responsible for RNA chain elongation and which exhibits RNA-dependent RNA polymerase activity (2, 5) and endonuclease activity (9, 16, 26). Previous research has already proved that the knockout of PR8 PB1-F2 reduced virus RNP activity, revealing that PR8 PB1-F2 contributes to virus RNP activity (27), even though PB1-F2 has no effect on the virus growth rate (40). In the present study, not only PR8 PB1-F2 but also H5N1 PB1-F2 and putative full-length swine-origin influenza A virus (S-OIV) PB1-F2 contributed to virus RNP activity. However, PR8 PB1-F2 and H5N1 PB1-F2 exhibit different biological behaviors, including different levels of expression, cellular localizations, and apoptosis enhancements. The molecular determinants of the different localizations were also addressed. The function of the putative PB1-F2 derived from S-OIV was also studied. The investigation described here reveals that PB1-F2 proteins derived from various viral strains exhibited distinct functions, possibly contributing to the variation in the virulence of influenza A viruses.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.     

Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.Influenza virus infections continue to represent a major public health threat. Epidemics caused by influenza A viruses (FLUAV) occur regularly, often leading to excess mortality in susceptible populations, and may result in devastating pandemics for humans (37). An avian FLUAV originating from Asia and currently circulating among domestic birds in many countries has the potential to infect and kill people. If further adaptation to humans occurs, this virus strain might become the origin of a future pandemic (57). Although influenza viruses are well characterized, the molecular determinants governing cross-species adaptation and enhanced virulence of emerging virus strains in humans are presently not well understood. The known viral virulence factors are the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), the nonstructural proteins NS1 and PB1-F2, and the polymerase complex. HA and NA are of key importance for host specificity and virulence because they determine specific receptor usage and efficient cell entry, as well as formation and release of progeny virus particles. NS1 is a multifunctional protein with interferon-antagonistic activity able to suppress host innate immune responses (11, 15). The small proapoptotic protein PB1-F2 induces more-severe pulmonary immunopathology and increases susceptibility to secondary bacterial pneumonia (3, 30). Recent evidence indicates that the polymerase complex consisting of the three subunits PA, PB1, and PB2 is also a determinant of virulence. Analyses of the 1918 pandemic virus showed that PB1 contributed to the high virulence of this deadly strain (38, 54, 56). Likewise, PB1 also contributed to the unusually high virulence of the pandemic viruses of 1957 and 1968 (23, 47). Interestingly, in recent avian-to-human transmissions of H5N1 and H7N7 viruses, the PB2 subunit was found to play a critical role (32, 40). Molecular studies revealed that an E-to-K exchange at position 627 of PB2 facilitates efficient replication of avian viruses in human cells (24, 33) and determines pathogenicity in mammals (18, 32, 51). Furthermore, recent analyses of highly pathogenic H5N1 viruses demonstrated that PA is involved in high virulence of these avian strains for both avian and mammalian hosts (21, 27).Moderately pathogenic FLUAV strains can be rendered more pathogenic by repeated passages in experimentally infected animals (2, 13, 16, 49, 55). During such adaptations, the evolving viruses frequently seem to acquire virulence-enhancing mutations in the polymerase genes. We recently characterized a virus pair with strikingly different virulences in mice and showed that the virulence-enhancing mutations of the highly virulent strain mapped to the HA, NA, and polymerase genes (13). The two A/Puerto Rico/8/34 (A/PR/8/34) strains are referred to here as high-virulence A/PR/8/34 (hvPR8) and low-virulence A/PR/8/34 (lvPR8). Interestingly, hvPR8 is also highly virulent in mice that carry functional alleles of the Mx1 resistance gene (17), most likely because it replicates rapidly enough to evade the innate immune response of naïve hosts (13).Here, we systematically analyzed which mutations in the three viral polymerase genes contribute to enhanced virulence of hvPR8. We found that two conservative mutations, one in PB2 (I504V) and one in PA (I550L), account for the high-virulence phenotype and that each single mutation considerably increases the activity of the reconstituted polymerase complex. Interestingly, in a new mouse adaptation experiment, the same I504V mutation in PB2 was acquired again by a highly virulent isolate as the only change in the polymerase complex. In contrast, another virulent, mouse-adapted isolate acquired two different mutations in PA and PB1. In this case, the change in PA had a greater impact on both enhanced polymerase activity and enhanced virulence than the mutation in PB1. These data demonstrate that increased polymerase activity contributes to high virus virulence and that human FLUAV have a range of options to achieve this goal.(This work was conducted by Thierry Rolling, Iris Koerner, and Petra Zimmermann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty [T.R.] or a Ph.D. degree from the Faculty of Biology [I.K. and P.Z.] of the University of Freiburg, Germany.)     

The Effects of Influenza A Virus PB1-F2 Protein on Polymerase Activity Are Strain Specific and Do Not Impact Pathogenesis

The influenza A virus PB1-F2 protein has been implicated as a virulence factor, but the mechanism by which it enhances pathogenicity is not understood. The PB1 gene segment of the H1N1 swine-origin influenza virus pandemic strain codes for a truncated PB1-F2 protein which terminates after 11 amino acids but could acquire the full-length form by mutation or reassortment. It is therefore important to understand the function and impact of this protein. We systematically assessed the effect that PB1-F2 expression has on viral polymerase activity, accumulation and localization of PB1, and replication in vitro and in mice. We used both the laboratory strain PR8 and a set of viruses engineered to study clinically relevant PB1-F2 proteins. PB1-F2 expression had modest effects on polymerase activity, PB1 accumulation, and replication that were cell type and virus strain dependent. Disruption of the PB1-F2 reading frame in a recent, seasonal H3N2 influenza virus strain did not affect these parameters, suggesting that this is not a universal function of the protein. Disruption of PB1-F2 expression in several backgrounds or expression of PB1-F2 from the 1918 pandemic strain or a 1956 H1N1 strain had no effect on viral lung loads in mice. Alternate mechanisms besides alterations to replication are likely responsible for the enhanced virulence in mammalian hosts attributed to PB1-F2 in previous studies.Seasonal influenza is responsible for significant morbidity and mortality worldwide. In the 1990s, it was estimated to kill 36,000 persons annually in the United States alone and 250,000 to 500,000 persons in the developed world, although hospitalization rates and mortality figures varied considerably from season to season based on the circulating strains (19, 20). Influenza A viruses also have the capability to cause a pandemic if they are sufficiently novel. Strains may emerge whole or in part from animal reservoirs and establish long-term (years to decades) zoonotic lineages in humans (23). The most striking example of this phenomenon occurred in 1918, when an avian virus of the H1N1 subtype crossed the species barrier and established related lineages in two mammalian hosts, swine and humans (16). This pandemic is thought to have killed more than 40 million persons worldwide. In 2009, a novel H1N1 influenza virus of swine origin (H1N1 S-OIV) emerged and is now causing the first pandemic the world has seen in more than 40 years (14). Because of the history of pandemic influenza and the current circulation of a novel pandemic strain, there is intense interest and urgency in understanding viral factors that allow expression of disease in humans.One such virulence factor is the influenza A virus protein PB1-F2 (8). This small (87 to 90 amino acids), 11th gene product was discovered in 2001 in a search for CD8⁺ epitopes in alternative reading frames of influenza A virus genes (2). It is encoded in the +1 reading frame of the PB1 gene segment and is translated from an AUG codon downstream of the PB1 start site, probably accessed through leaky ribosomal scanning. It has been shown to contribute to virulence both directly and indirectly, through modulation of responses to bacteria (3, 11). The exact mechanism(s) through which virulence is increased by PB1-F2 expression, however, is not yet understood. Three effects of PB1-F2 expression have been suggested so far. It has been demonstrated to cause cell death in some cell types (2, 5), it has been shown to induce inflammation by recruitment of inflammatory cells in mice (11), and it has been determined to bind PB1 and to increase the activity of the influenza virus polymerase in vitro (10).The function of the PB1-F2 protein in the life cycle of influenza virus is as unclear as its precise role in virulence. Given that almost all avian influenza virus strains express a full-length PB1-F2 protein (27), it is likely to contribute to survival or transmission in the natural avian host. After introduction of viruses into mammalian hosts such as humans or swine, however, the protein often becomes truncated during adaptation, implying that any effects it might induce are not necessary for virus viability and transmission in these hosts. The 1918 H1N1 virus had a full-length PB1-F2 protein, which has been demonstrated to contribute to virulence in mice (3, 11). During the evolution of H1N1 viruses in humans over time, a stop codon at position 58 in the PB1-F2 amino acid sequence appeared around 1950 and has been retained in the human H1N1 lineage since its reemergence in 1977. Similarly, multiple swine lineages of influenza A virus have had truncations appear at different positions, including position 58, such that 25% of swine PB1-F2 sequences in GenBank lack the C-terminal portion of the protein (27). The H3N2 lineage of viruses in humans has retained a full-length PB1-F2 protein since the introduction of a new PB1 gene segment during the 1968 pandemic, although considerable variation in sequence has occurred during evolution since that time. It is tempting to map these differences in PB1-F2 expression onto patterns of human excess mortality over time, since higher mortality was associated with H1N1 epidemics in the 1930s and 1940s than has been seen since and more excess mortality occurred in recent years with H3N2 viruses than with either H1N1 or influenza B viruses (reviewed in reference 12). Differences in primary virulence or the association with bacteria mediated by PB1-F2 expression could be at least partly responsible for these observed epidemiologic trends.A recent paper from Wise et al. has shown that a 12th influenza A virus gene product, N40, is also expressed from the PB1 gene segment (24). A delicate balance between PB1, PB1-F2, and N40 appears to be in place. Polymerase activity measured by an in vitro assay was affected by changes in this balance, suggesting a potential importance for replication. If these differences translate to differences in replication, then this could be a key factor in virulence in the host. However, to this point, most studies have utilized a single laboratory variant of influenza A virus, A/Puerto Rico/8/34 (H1N1; PR8), in a limited set of cell types, in assays performed in vitro. We undertook this study to determine the relevance of potential changes in replication mediated by PB1-F2 expression, utilizing several different epidemiologically important virus strains. We found that the effects on polymerase activity and in vitro replication efficiency were virus and cell type specific and did not mediate changes in viral lung load in animals.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.Influenza A virus is a highly infectious respiratory pathogen, causing 3 to 5 million severe cases yearly while the recent H1N1 pandemic has spread to over 200 countries and resulted in over 15,000 WHO-confirmed deaths since its emergence in March 2009 (57). Influenza virus particles are enveloped structures that contain nine identified viral polypeptides. The lipid envelope is derived by budding from the apical plasma membrane and contains the viral integral membrane proteins hemagglutinin (HA) and neuraminidase (NA) as well as the M2 ion channel. Internally, virus particles contain a matrix protein (M1), small quantities of the NS2/NEP polypeptide, and eight genomic segments of negative-sense RNA that are separately encapsidated into ribonucleoprotein (RNP) particles by the viral nucleoprotein (NP) and tripartite polymerase complex (PB1, PB2, and PA). M1 is thought to form a link between the RNPs and the cytoplasmic tails of the viral membrane proteins though M2 may also play a role (39). The minimal viral protein requirements for budding are disputed; while initial studies suggested that M1 was the main driver of budding (21, 34), more recent work proposes that the glycoproteins HA and NA are responsible (8).Further complicating the analysis of influenza A virus budding is the observation that most strains of the virus form two distinct types of virions: spherical particles approximately 100 nm in diameter and much longer filamentous particles up to 30 μm in length (38). Of the viral proteins, M1 is the primary determinant of particle shape (3, 17) although other virus genes also play a role. It is also likely that host factors are involved in the process as cells with fully differentiated apical and basolateral membranes produce more filaments than nonpolarized cell types (42). While it is tempting to speculate that virus morphology and budding are regulated by the same cellular process, the fact that spherical budding occurs in the absence of an intact actin cytoskeleton while filament formation does not (42, 48) indicates some level of divergence in the mechanisms responsible for spherical and filamentous virion morphogenesis.The means by which viral and cellular membranes are separated are also unclear. Unlike many other enveloped viruses, including retroviruses (19, 36, 52) and herpes simplex virus (12), influenza A virus does not utilize the cellular endosomal sorting complex required for transport (ESCRT) pathway (5, 8). However, recent reports indicate that some viruses, including human cytomegalovirus (HCMV) (32), the hantavirus Andes virus (44), and respiratory syncytial virus (RSV) may employ a Rab11-mediated pathway during assembly and/or budding (4, 51). The Rab family of small GTPases is involved in targeting vesicle trafficking, mediating a wide range of downstream processes including endosomal trafficking and membrane fusion/fission events (reviewed in references 53 and 58). Rab11 is involved in trafficking proteins and vesicles between the trans-Golgi network (TGN), recycling endosome, and the plasma membrane (9, 49, 50) as well as playing a role in actin remodeling, cytokinesis, and abscission (27, 41, 55). Apical recycling endosome (ARE) trafficking is of particular interest in the context of viral infection as other negative-sense RNA viruses have been shown to assemble and/or traffic virion components through the ARE prior to final assembly and budding at the plasma membrane (4, 44, 51). Rab11 function is modulated and targeted through interactions with Rab11 family interacting proteins (Rab11-FIPs) that direct it to specific subcellular locations (23, 25, 26) by binding to actin or microtubule-based motor proteins (24, 26, 47). While Rab11-FIPs recognize both isoforms of Rab11 (a and b [Rab11a/b]) through a conserved amphipathic α-helical motif, they differ in their ability to bind either the GTP-bound form of Rab11 (FIP1, FIP3, FIP4, and Rip11) or both the GTP and GDP-bound forms (FIP2) (23, 30). FIP1 and FIP2 have been implicated in RSV budding (4, 51) while FIP4 is important for trafficking of HCMV components (32). FIP3 has not previously been linked with virus budding but plays an important role in both cell motility and cytokinesis, regulating actin dynamics and endosomal membrane trafficking (29, 55).In light of the normal cellular functions of Rab11 and its effectors and of their reported involvement in the budding of other viruses, we examined the role of this cellular pathway in influenza virus budding. We find that Rab11-FIP3 is essential for filamentous but not spherical virion formation while Rab11 is required for both forms of virus budding.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose = 2.6 log₁₀ 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA_97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA_97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.Migratory waterfowl are the natural reservoir of influenza A viruses (11, 53). The viruses replicate efficiently in their natural hosts but replicate poorly if at all in other species (53). However, these viruses can undergo adaptation or genetic reassortment to infect other hosts (43, 44, 53), including humans. Since 1997, the World Health Organization has documented more than 400 laboratory-confirmed cases of human infection with H5N1 avian influenza virus (54).The molecular basis of influenza virus host range restriction and adaptation to a new host species is poorly understood. Mutations associated with cross-species adaptation are thought to be associated with increased virulence (30). Therefore, studies in animal models have attempted to identify the viral molecular determinants of virulence in specific hosts. Reverse genetics (Rg) methods have also identified genetic differences that affect virus virulence and host range, including changes in the viral internal proteins. Experimental infection of mouse lungs is an effective approach for understanding influenza virus virulence and adaptation (reviewed by A. C. Ward [51]). To acquire virulence in mice, influenza A viruses usually must adapt to these hosts over several consecutive generations (serial passages) in the lungs or brain (1, 25, 30). Previous studies have found that the acquisition of virulence during adaptation in the mouse model is associated with mutations in the HA, NP, NA, M, and NS genes and one or more polymerase genes (2, 3, 18, 36, 42, 51). The polymerase basic protein 2 (PB2) gene is a particularly well-characterized polymerase subunit (7, 23, 40, 46). The PB1 and polymerase acidic protein (PA) genes have been implicated in mouse lung virulence (5, 18, 36, 39, 49) but have shown no evidence of having acquired mutations during adaptation (52). However, the many studies conducted to date have focused mainly on highly pathogenic avian influenza (HPAI) viruses such as the H1N1, H5N1, and H7N7 subtypes (7, 23, 48, 50).Various low-pathogenic avian influenza (LPAI) viruses are considered to be potential genetic contributors to the next pandemic strain. Lee et al. (2009) recently reported the presence of avian-like LPAI H5N2 viruses in a number of Korean swine and proposed that the efficient transmissibility of the swine-adapted H5N2 virus could facilitate spread of the virus. They suggested that this adapted virus could potentially serve as a model for pandemic outbreaks of HPAI (e.g., H5N1 and H7N7) virus or could become a pandemic strain itself (21). These findings prompted our interest in the adaptation of an LPAI virus often harbored by wild migratory birds of South Korea. In our ongoing surveillance from 2004 to 2008, approximately 27% of the viruses isolated were of the H5N2 subtype (unpublished data). Studies show that influenza viruses with different genetic backgrounds can acquire different mutations during adaptation in mice. Therefore, we sought to determine whether this common H5N2 virus (nonlethal in mice) would undergo changes different from those observed in highly virulent viruses during adaptation in mice. Wild-bird influenza virus strain A/Aquatic bird/Korea/W81/05 (W81) was adapted in mice over 11 passages and became highly virulent. To identify molecular determinants of this adaptation and altered virulence, we used Rg-generated recombinant viruses to compare the parental and mouse-adapted strains. Here we show that the PA subunit of the polymerase complex, independently of PB2, contributed to adaptation and increased virulence in our mammalian model.