Bacteriocins Revitalize Non-Effective Penicillin G to Overcome Methicillin-Resistant Staphylococcus pseudintermedius.

The rise of antibiotic-resistant bacteria is among the biggest challenges in human and veterinary medicine. One of the major factors that contributes to resistance is use of frontline clinical antibiotics in veterinary practices. To avoid this problem, searching for antimicrobials aimed at veterinary applications is becoming especially important. Thiopeptide micrococcin P1 and leaderless peptide EntEJ97s are two different bacteriocins that are very active against many gram-positive bacteria; however, sensitive bacteria can rapidly develop resistance towards those bacteriocins. To overcome this problem, we searched for synergy between those bacteriocins and conventional antibiotics against methicillin-resistant Staphylococcus pseudintermedius (MRSP): a common pathogen in animal skin infections. The two bacteriocins acted synergistically with each other and with penicillin G against MRSP clinical isolates in both planktonic and biofilm assays; they also prevented resistance development. The therapeutic potential was further validated in a murine skin infection model that showed that a combination of micrococcin P1, EntEJ97s and penicillin G reduced cell-forming units of MRSP by 2-log10 CFU/g. Taken together, our data show that a combination of bacteriocins with conventional antibiotics can not only prevent resistance development but also pave the way to revitalize some old, less useful antibiotics, such as penicillin, which by itself has no effect on methicillin-resistant pathogens.

A Strong Synergy Between the Thiopeptide Bacteriocin Micrococcin P1 and Rifampicin Against MRSA in a Murine Skin Infection Model.

Antibiotic-resistant bacterial pathogens have become a serious threat worldwide. One of these pathogens is methicillin-resistant Staphylococcus aureus (MRSA), a major cause of skin and soft tissue infections. In this study we identified a strain of Staphylococcus equorum producing a substance with high antimicrobial activity against many Gram-positive bacteria, including MRSA. By mass spectrometry and whole genome sequencing the antimicrobial substance was identified as the thiopeptide bacteriocin micrococcin P1 (MP1). Based on its properties we developed a one-step purification protocol resulting in high yield (15 mg/L) and high purity (98%) of MP1. For shorter incubation times (5-7 h) MP1 was very potent against MRSA but the inhibitory effect was overshadowed by resistance development during longer incubation time (24h or more). To overcome this problem a synergy study was performed with a number of commercially available antibiotics. Among the antibiotics tested, the combination of MP1 and rifampicin gave the best synergistic effect, with MIC values 25 and 60 times lower than for the individual drugs, respectively. To assess the therapeutic potential of the MP1-rifampicin combination, we used a murine skin infection model based on the use of the multidrug-resistant luciferase-tagged MRSA strain Xen31. As expected, neither of the single antimicrobials (MP1 or rifampicin) could eradicate Xen31 from the wounds. By contrary, the MP1-rifampicin combination was efficient not only to eradicate but also to prevent the recurrence of Xen31 infection. Furthermore, compared to fucidin cream, which is commonly used in skin infection treatments, MP1-rifampicin combination was superior in terms of preventing resistance development. Our results show that combining MP1, and probably other thiopeptides, with antibiotics can be a promising strategy to treat SSTIs caused by MRSA and likely many other Gram-positive bacteria.

A bacteriocin-based treatment option for Staphylococcus haemolyticus biofilms.

Bacteriocins are ribosomally-synthesized antimicrobial peptides, showing great potential as novel treatment options for multidrug-resistant pathogens. In this study, we designed a novel hybrid bacteriocin, Hybrid 1 (H1), by combing the N-terminal part and the C-terminal part of the related bacteriocins enterocin K1 (K1) and enterocin EJ97 (EJ97), respectively. Like the parental bacteriocins, H1 used the membrane-bound protease RseP as receptor, however, it differed from the others in the inhibition spectrum. Most notably, H1 showed a superior antimicrobial effect towards Staphylococcus haemolyticus-an important nosocomial pathogen. To avoid strain-dependency, we further evaluated H1 against 27 clinical and commensal S. haemolyticus strains, with H1 indeed showing high activity towards all strains. To curtail the rise of resistant mutants and further explore the potential of H1 as a therapeutic agent, we designed a bacteriocin-based formulation where H1 was used in combination with the broad-spectrum bacteriocins micrococcin P1 and garvicin KS. Unlike the individual bacteriocins, the three-component combination was highly effective against planktonic cells and completely eradicated biofilm-associated S. haemolyticus cells in vitro. Most importantly, the formulation efficiently prevented development of resistant mutants as well. These findings indicate the potential of a bacteriocins-based formulation as a treatment option for S. haemolyticus.

Staphylococcal Biofilms: Challenges and Novel Therapeutic Perspectives.

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms-three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches.

A bacteriocin-based antimicrobial formulation to effectively disrupt the cell viability of methicillin-resistant Staphylococcus aureus (MRSA) biofilms.

Antibiotic-resistant and biofilm-associated infections brought about by methicillin-resistant Staphylococcus aureus (MRSA) strains is a pressing issue both inside as well as outside nosocomial environments worldwide. Here, we show that a combination of two bacteriocins with distinct structural and functional characteristics, garvicin KS, and micrococcin P1, showed a synergetic antibacterial activity against biofilms produced in vitro by S. aureus, including several MRSA strains. In addition, this bacteriocin-based antimicrobial combination showed the ability to restore the sensitivity of the highly resilient MRSA strain ATCC 33591 to the ß-lactam antibiotic penicillin G. By using a combination of bacterial cell metabolic assays, confocal and scanning electron microscopy, we show that the combination between garvicin KS, micrococcin P1, and penicillin G potently inhibit cell viability within S. aureus biofilms by causing severe cell damage. Together these data indicate that bacteriocins can be valuable therapeutic tools in the fight against biofilm-associated MRSA infections.

Successful Development of Bacteriocins into Therapeutic Formulation for Treatment of MRSA Skin Infection in a Murine Model.

The emergence of antibiotic-resistant pathogens has caused a serious worldwide problem in infection treatment in recent years. One of the pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which is a major cause of skin and soft tissue infections. Alternative strategies and novel sources of antimicrobials to solve antibiotic resistance problems are urgently needed. In this study, we explored the potential of two broad-spectrum bacteriocins, garvicin KS and micrococcin P1, in skin infection treatments. The two bacteriocins acted synergistically with each other and with penicillin G in killing MRSA in vitro The MICs of the antimicrobials in the three-component mixture were 40 ng/ml for micrococcin P1 and 2 µg/ml for garvicin KS and penicillin G, which were 62, 16, and at least 1,250 times lower than their MICs when assessed individually. To assess its therapeutic potential further, we challenged the three-component formulation in a murine skin infection model with the multidrug-resistant luciferase-tagged MRSA Xen31, a strain derived from the clinical isolate S. aureus ATCC 33591. Using the tagged-luciferase activity as a reporter for the presence of Xen31 in wounds, we demonstrated that the three-component formulation was efficient in eradicating the pathogen from treated wounds. Furthermore, compared to Fucidin cream, which is an antibiotic commonly used in skin infection treatments, our formulation was also superior in terms of preventing resistance development.

Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance.

Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance.

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self-regenerating multi-layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long-term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV-16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV-16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV-16-positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high-risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non-HPV-associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Human papillomavirus infection and induction of neoplasia: a matter of fitness.

The aetiologic association between infection with certain human papillomavirus (HPV) types, high-grade squamous neoplasia, and cancer at different epithelial sites is well established. In this review we briefly discuss recent breakthroughs in the regulation of squamous epithelia in homeostasis and disease, and provide a view of how these discoveries modify our understanding of how HPV-induced neoplasia in squamous epithelia is triggered. Taken together, these observations highlight how HPVs have evolved the ability to inactivate the products of genes that are frequently mutated in non-HPV-associated pre-neoplasia and squamous cell carcinoma of sun-exposed skin, and introduce a Darwinian model of clonal evolution of HPV-infected cells. These concepts are considered against our current understanding of transformation zones where HPV-associated cancers occur more frequently, and other sites of non-productive (or abortive) HPV infection.

Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways.

A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.

The high-risk HPV E6 target scribble (hScrib) is required for HPV E6 expression in cervical tumour-derived cell lines.

The ability of high-risk HPV E6 oncoproteins to target cellular proteins which harbor PDZ domains is believed to play an important role in the virus life cycle and to influence the ability of these viruses to bring about malignant transformation. Whilst many of these PDZ proteins are potential tumour suppressors, involved in the control of cell polarity and cell-contact, recent studies suggest that mislocalisation or overexpression might result in the emergence of oncogenic functions. This has been shown most clearly for two E6 targets, hDlg and hScrib. In this study we show that hScrib plays such a role in HeLa cells, where its expression is required for maintaining high levels of HPV-18 E6 protein. Loss of hScrib has no effect on E6 stability but results in lower levels of E6 transcription and a reduced rate of E6 translation. We further show that, in the context of cervical tumour-derived cell lines, both hScrib and E6 cooperate in the activation of the S6 kinase signaling pathway, and thereby contribute towards maintaining high rates of protein translation. These results indicate that the residual hScrib that is present within HPV transformed cells is pro-oncogenic, and highlights the dual functions of E6 cell polarity targets.

HPV-16 impairs the subcellular distribution and levels of expression of protein phosphatase 1γ in cervical malignancy.

BACKGROUND: The high risk Human Papillomavirus (HPV) E6 oncoproteins play an essential role in the development of cervical malignancy. Important cellular targets of E6 include p53 and the PDZ domain containing substrates such as hScrib and Dlg. We recently showed that hScrib activity was mediated in part through recruitment of protein phosphatase 1γ (PP1γ). METHODS: Expression patterns of hScrib and PP1γ were assessed by immunohistochemistry of HPV-16 positive cervical intraepithelial neoplasm (CIN), classified as CIN1 (n = 4), CIN2 (n = 8), CIN3 (n = 8), cervical carcinoma tissues (n = 11), and HPV-negative cervical tissues (n = 8), as well as by subfractionation assay of the HPV-16 positive cervical cancer cell lines, CaSki and SiHa. To explore the effects of the HPV-16 oncoproteins, we have performed siRNA knockdown of E6/E7 expression, and monitored the effects on the expression patterns of hScrib and PP1γ. RESULTS: We show that PP1γ levels in HPV-16 positive tumour cells are reduced in an E6/E7 dependent manner. Residual PP1γ in these cells is found mostly in the cytoplasm as opposed to the nucleus where it is predominantly found in normal cells. We have found a striking concordance with redistribution in the pattern of expression (9/11; 81.8%) and loss of PP1γ expression in HPV-16 positive cervical tumours (2/11; 18.2%). Furthermore, this loss of PP1γ expression and redistribution in the pattern of expression occurs progressively as the lesions develop (8/8; 100%). CONCLUSION: Together, these results suggest that PP1γ may be a novel target of the HPV-16 oncoproteins and indicate that it might be a potential novel biomarker for HPV-16 induced malignancy.

Human papillomavirus molecular biology and disease association.

Human papillomaviruses (HPVs) have evolved over millions of years to propagate themselves in a range of different animal species including humans. Viruses that have co-evolved slowly in this way typically cause chronic inapparent infections, with virion production in the absence of apparent disease. This is the case for many Beta and Gamma HPV types. The Alpha papillomavirus types have however evolved immunoevasion strategies that allow them to cause persistent visible papillomas. These viruses activate the cell cycle as the infected epithelial cell differentiates in order to create a replication competent environment that allows viral genome amplification and packaging into infectious particles. This is mediated by the viral E6, E7, and E5 proteins. High-risk E6 and E7 proteins differ from their low-risk counterparts however in being able to drive cell cycle entry in the upper epithelial layers and also to stimulate cell proliferation in the basal and parabasal layers. Deregulated expression of these cell cycle regulators underlies neoplasia and the eventual progression to cancer in individuals who cannot resolve high-risk HPV infection. Most work to date has focused on the study of high-risk HPV types such as HPV 16 and 18, which has led to an understanding of the molecular pathways subverted by these viruses. Such approaches will lead to the development of better strategies for disease treatment, including targeted antivirals and immunotherapeutics. Priorities are now focused toward understanding HPV neoplasias at sites other than the cervix (e.g. tonsils, other transformation zones) and toward understanding the mechanisms by which low-risk HPV types can sometimes give rise to papillomatosis and under certain situations even cancers.

Restoration of MAGI-1 expression in human papillomavirus-positive tumor cells induces cell growth arrest and apoptosis.

UNLABELLED: The cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins target a number of cellular proteins that contain PDZ domains. However, the role of many of these interactions in either the HPV life cycle or in HPV-induced malignancy remains to be defined. Previous studies had shown that MAGI-1 was one of the most strongly bound PDZ domain-containing substrates of E6, and one consequence of this interaction appeared to facilitate the perturbation of tight junctions (TJs) by E6. In this study, we describe the generation of a mutation, K499E, within the MAGI-1 PDZ1 domain, which is resistant to E6 targeting. This mutant allows restoration of MAGI-1 expression in HPV-positive cells and defines additional activities of MAGI-1 that are overcome as a consequence of the association with E6. The reexpression of MAGI-1 in HPV-positive cells results in an increased recruitment of ZO-1 and PAR3 to sites of cell-cell contact, repression of cell proliferation, and induction of apoptosis. While the K499E mutation does not significantly affect these intrinsic activities of MAGI-1 in HPV-negative cells, its resistance to E6 targeting in an HPV-positive setting results in more cells expressing the mutant MAGI-1 than the wild-type MAGI-1, with a corresponding increase in TJ assembly, induction of apoptosis, and reduction in cell proliferation. These studies provide compelling evidence of a direct role for the perturbation of MAGI-1 function by E6 in the HPV life cycle and in HPV-induced malignancy. IMPORTANCE: It is clear that the targeting of PDZ-containing substrates by E6 is important for the normal viral life cycle and for the progression to malignancy. Nevertheless, which of these PDZ domain-containing proteins is relevant for HPV pathology is still elusive. In a previous study, we provided evidence that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins; however, the biological consequences associated with loss of MAGI-1 expression in HPV-positive cervical cancer cells are still poorly understood. Using a mutant MAGI-1, resistant to E6-mediated degradation, we show that its expression in cervical cancer cells promotes membrane recruitment of the tight junction-associated proteins ZO-1 and PAR3, represses cell proliferation, and promotes apoptosis. These findings suggest that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis.

A novel interaction between hScrib and PP1γ downregulates ERK signaling and suppresses oncogene-induced cell transformation.

Previous studies have shown that the cell polarity regulator hScrib interacts with, and consequently controls, the ERK signaling pathway. This interaction occurs through two well-conserved Kinase Interacting Motifs, which allow hScrib to bind ERK1 directly, resulting in a reduction in the levels of phospho-ERK. This suggests that hScrib might recruit a phosphatase to regulate this signaling pathway. Using a proteomic approach we now show that Protein Phosphatase 1γ (PP1γ) is a major interacting partner of hScrib. This interaction is direct and occurs through a conserved PP1γ interaction motif on the hScrib protein, and this interaction appears to be required for hScrib's ability to downregulate ERK phosphorylation. In addition, hScrib also controls the pattern of PP1γ localization, where loss of hScrib enhances the nuclear translocation of PP1γ. Furthermore, we also show that the ability of hScrib to interact with PP1γ is important for the ability of hScrib to suppress oncogene-induced transformation of primary rodent cells. Taken together, these results demonstrate that hScrib acts as a scaffold to integrate the control of the PP1γ and ERK signaling pathways and explains how disruption of hScrib localisation can contribute towards the development of human malignancy.

Human papillomaviruses and the specificity of PDZ domain targeting.

The human papillomavirus (HPV) E6 oncoprotein is fundamental to the ability of these viruses to induce human malignancy. A defining characteristic of the HPV E6 oncoproteins found in cancer-causing HPV types is the presence of a PDZ binding motif at their extreme C-terminus. Through this motif, E6 is able to interact with a large number of cellular proteins that contain PDZ domains. Many of these cellular proteins are involved in regulation of processes associated with the control of cell attachment, cell proliferation, cell polarity and cell signaling. How E6 targets multiple proteins containing the same recognition domain is still an open question. In this review, we highlight aspects of E6 function and biology that help to answer this question, and thereby provide insight into the role of these substrates during development of HPV-induced malignancy.

PDZ domains: the building blocks regulating tumorigenesis.

Over 250 PDZ (PSD95/Dlg/ZO-1) domain-containing proteins have been described in the human proteome. As many of these possess multiple PDZ domains, the potential combinations of associations with proteins that possess PBMs (PDZ-binding motifs) are vast. However, PDZ domain recognition is a highly specific process, and much less promiscuous than originally thought. Furthermore, a large number of PDZ domain-containing proteins have been linked directly to the control of processes whose loss, or inappropriate activation, contribute to the development of human malignancies. These regulate processes as diverse as cytoskeletal organization, cell polarity, cell proliferation and many signal transduction pathways. In the present review, we discuss how PBM-PDZ recognition and imbalances therein can perturb cellular homoeostasis and ultimately contribute to malignant progression.

Stabilization of HPV16 E6 protein by PDZ proteins, and potential implications for genome maintenance.

The E6 protein from high-risk human papillomaviruses appears necessary for persistence of viral episomes in cells but the underlying mechanism is unclear. E6 has many activities, including its ability to bind and degrade PDZ domain-containing proteins, such as hScrib. However little is known about the role of these interactions for E6 function and the viral life cycle. We now show that the levels of expression of wild-type E6 are increased in the presence of hScrib whilst a mutant E6 protein lacking the PDZ-binding motif is found at lower levels as it is turned over more rapidly by the proteasome. This correlates with an inability of genomes containing this mutation to be maintained as episomes. These results show that E6 association with certain PDZ domain-containing proteins can stabilize the levels of E6 expression and provides one explanation as to how the PDZ-binding capacity of E6 might contribute to genome episomal maintenance.

Analysis of the PDZ binding specificities of Influenza A virus NS1 proteins.

The Influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant.In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.