Nuclease Activity of the Junín Virus Nucleoprotein C-Terminal Domain.

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen.

Assembly of the Tripartite and RNA Condensates of the Respiratory Syncytial Virus Factory Proteins In Vitro: Role of the Transcription Antiterminator M2-1.

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid-liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein-RNA coacervates based on the amphiphilic behavior of M2-1 and finely tuned by stoichiometry. M2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M2-1 behaves differently in the protein phase as opposed to the protein-RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

Biomolecular Condensation of the Human Papillomavirus E2 Master Regulator with p53: Implications in Viral Replication.

p53 exerts its tumour suppressor activity by modulating hundreds of genes and it can also repress viral replication. Such is the case of human papillomavirus (HPV) through targeting the E2 master regulator, but the biochemical mechanism is not known. We show that the C-terminal DNA binding domain of HPV16 E2 protein (E2C) triggers heterotypic condensation with p53 at a precise 2/1 E2C/p53 stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Interestingly, transfection experiments show that E2 co-localizes with p53 in the nucleus with a grainy pattern, and recruits p53 to chromatin-associated foci, a function independent of the DNA binding capacity of p53 as judged by a DNA binding impaired mutant. Depending on the length, DNA can either completely dissolve or reshape heterotypic droplets into irregular condensates containing p53, E2C, and DNA, and reminiscent of that observed linked to chromatin. We propose that p53 is a scaffold for condensation in line with its structural and functional features, in particular as a promiscuous hub that binds multiple cellular proteins. E2 appears as both client and modulator, likely based on its homodimeric DNA binding nature. Our results, in line with the known role of condensation in eukaryotic gene enhancement and silencing, point at biomolecular condensation of E2 with p53 as a means to modulate HPV gene function, strictly dependent on host cell replication and transcription machinery.

Conformational buffering underlies functional selection in intrinsically disordered protein regions.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

Deconstructing virus condensation.

Viruses have evolved precise mechanisms for using the cellular physiological pathways for their perpetuation. These virus-driven biochemical events must be separated in space and time from those of the host cell. In recent years, granular structures, known for over a century for rabies virus, were shown to host viral gene function and were named using terms such as viroplasms, replication sites, inclusion bodies, or viral factories (VFs). More recently, these VFs were shown to be liquid-like, sharing properties with membrane-less organelles driven by liquid-liquid phase separation (LLPS) in a process widely referred to as biomolecular condensation. Some of the best described examples of these structures come from negative stranded RNA viruses, where micrometer size VFs are formed toward the end of the infectious cycle. We here discuss some basic principles of LLPS in connection with several examples of VFs and propose a view, which integrates viral replication mechanisms with the biochemistry underlying liquid-like organelles. In this view, viral protein and RNA components gradually accumulate up to a critical point during infection where phase separation is triggered. This yields an increase in transcription that leads in turn to increased translation and a consequent growth of initially formed condensates. According to chemical principles behind phase separation, an increase in the concentration of components increases the size of the condensate. A positive feedback cycle would thus generate in which crucial components, in particular nucleoproteins and viral polymerases, reach their highest levels required for genome replication. Progress in understanding viral biomolecular condensation leads to exploration of novel therapeutics. Furthermore, it provides insights into the fundamentals of phase separation in the regulation of cellular gene function given that virus replication and transcription, in particular those requiring host polymerases, are governed by the same biochemical principles.

The structure of the extended E2 DNA-binding domain of the bovine papillomavirus-1.

Bovine papillomavirus proteins were extensively studied as a prototype for the human papillomavirus. Here, the crystal structure of the extended E2 DNA-binding domain of the dominant transcription regulator from the bovine papillomavirus strain 1 is described in the space group P31 21. We found two protein functional dimers packed in the asymmetric unit. This new protein arrangement inside the crystal led to the reduction of the mobility of a previously unobserved loop directly involved in the protein-DNA interaction, which was then modeled for the first time.

Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus.

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main ß-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.

Topology Dictates Evolution of Regulatory Cysteines in a Family of Viral Oncoproteins.

Redox regulation in biology is largely operated by cysteine chemistry in response to a variety of cell environmental and intracellular stimuli. The high chemical reactivity of cysteines determines their conservation in functional roles, but their presence can also result in harmful oxidation limiting their general use by proteins. Papillomaviruses constitute a unique system for studying protein sequence evolution since there are hundreds of anciently evolved stable genomes. E7, the viral transforming factor, is a dimeric, cysteine-rich oncoprotein that shows both conserved structural and variable regulatory cysteines constituting an excellent model for uncovering the mechanism that drives the acquisition of redox-sensitive groups. By analyzing over 300 E7 sequences, we found that although noncanonical cysteines show no obvious sequence conservation pattern, they are nonrandomly distributed based on topological constrains. Regulatory residues are strictly excluded from six positions stabilizing the hydrophobic core while they are enriched in key positions located at the dimerization interface or around the Zn+2 ion. Oxidation of regulatory cysteines is linked to dimer dissociation, acting as a reversible redox-sensing mechanism that triggers a conformational switch. Based on comparative sequence analysis, molecular dynamics simulations and biophysical analysis, we propose a model in which the occurrence of cysteine-rich positions is dictated by topological constrains, providing an explanation to why a degenerate pattern of cysteines can be achieved in a family of homologs. Thus, topological principles should enable the possibility to identify hidden regulatory cysteines that are not accurately detected using sequence based methodology.

Mechanism of Tetramer Dissociation, Unfolding, and Oligomer Assembly of Pneumovirus M2-1 Transcription Antiterminators.

Among Mononegavirales, the Pneumovirus family stands out by its RNA polymerase processivity that relies on a transcription antiterminator, the M2-1 protein, which also plays a key role in viral particle assembly. Biophysical and structural evidence shows that this RNA-binding tetramer is strongly modulated by a CCCH Zn2+ binding motif. We show that while the global dissociation/unfolding free energy is 10 kcal mol-1, more stable for the respiratory syncytial virus M2-1, the human metapneumovirus (HMPV) counterpart shows a 7 kcal mol-1 higher intersubunit affinity. Removal of Zn2+ from both homologues leads to an apo-monomer of identical secondary structure that further undergoes a slow irreversible oligomerization. Mutation of the histidine residue of the Zn2+ motif to cysteine or alanine leads directly to large oligomers, strongly suggesting that metal coordination has an exquisite precision for modulating the quaternary arrangement. Zn2+ removal is very slow and requires subdenaturing concentrations of guanidine chloride, suggesting a likely local folding energy barrier. Exploring a broad combination of denaturant and ethylenediaminetetraacetic acid conditions, we showed that the metapneumovirus protein has to overcome a higher energy barrier to trigger Zn2+ removal-driven dissociation, in concordance with a slower dissociation kinetics. In silico modeling of open and close conformations for both M2-1 tetramers together with interaction energy calculations reveals that the gradual opening of protomers decreases the number of intersubunit contacts. Half of the interaction energy holding each protomer in the tetramer comes from the CCCH motif, while HMPV-M2-1 harbors additional contacts between the CCCH motif of one subunit and the core domain of a protomer located in trans, allowing the rationalization of the experimental data obtained. Overall, the evidence points at a key role of the CCCH motif in switching between structural and consequently functional alternatives of the M2-1 protein.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein.

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

Cooperative RNA Recognition by a Viral Transcription Antiterminator.

RNA transcription of mononegavirales decreases gradually from the 3' leader promoter toward the 5' end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M2-1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M2-1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M2-1. Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M2-1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30.

Structure and stability of the Human respiratory syncytial virus M2-1 RNA-binding core domain reveals a compact and cooperative folding unit.

Human syncytial respiratory virus is a nonsegmented negative-strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2-1. The puzzling mechanism of action of M2-1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA-binding activity is located in a monomeric globular `core' domain, a high-resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full-length M2-1 tetramer, with additional electron density for the C-terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two-state equilibrium. These results constitute a further step in the understanding of this unique RNA-binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target.

Hidden Structural Codes in Protein Intrinsic Disorder.

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and ß-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Regulation of Tacaribe Mammarenavirus Translation: Positive 5' and Negative 3' Elements and Role of Key Cellular Factors.

Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.

Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein.

Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn+2 binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism.

Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex.

Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring "late" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.

Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.

The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 µM). A large secondary structure change into ß-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive ß-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined.

Convergent evolution and mimicry of protein linear motifs in host-pathogen interactions.

Pathogen linear motif mimics are highly evolvable elements that facilitate rewiring of host protein interaction networks. Host linear motifs and pathogen mimics differ in sequence, leading to thermodynamic and structural differences in the resulting protein-protein interactions. Moreover, the functional output of a mimic depends on the motif and domain repertoire of the pathogen protein. Regulatory evolution mediated by linear motifs can be understood by measuring evolutionary rates, quantifying positive and negative selection and performing phylogenetic reconstructions of linear motif natural history. Convergent evolution of linear motif mimics is widespread among unrelated proteins from viral, prokaryotic and eukaryotic pathogens and can also take place within individual protein phylogenies. Statistics, biochemistry and laboratory models of infection link pathogen linear motifs to phenotypic traits such as tropism, virulence and oncogenicity. In vitro evolution experiments and analysis of natural sequences suggest that changes in linear motif composition underlie pathogen adaptation to a changing environment.

Argentophilic nucleolus organizer region as a proliferation marker in cervical intraepithelial neoplasia grade 1 of the uterine cervix.

AIM: p16INK4a and argentophilic nucleolus organizer region (AgNOR) can be used as markers for progression of cervical intraepithelial neoplasia grade 1 (CIN1) of the uterine cervix. Our objective was to study the predictive value of the AgNOR technique as a progression marker of CIN1 and its correlation with p16INK4A. MATERIAL AND METHODS: One uterine cervix biopsy from each of 75 patients with diagnosis of CIN1 was selected. All of these patients underwent a second biopsy, and these were also used for the study. RESULTS: The second biopsies showed: regression (20 patients), persistent CIN1 (38 patients), progression to CIN2 (10 patients) and progression to CIN3 (seven patients). p16INK4A showed reactivity in 67 of the 75 first CIN1 biopsies: 12 of the 20 cases that cleared the lesions and the 55 cases with persistent or progressive lesions were positive for p16INK4a (specificity: 40%; sensitivity: 100%; positive predictive value [PPV]: 82%; negative predictive value [NPV]: 100%). Samples with AgNOR areas less than 3.0 µ(2) returned in all cases, but patients whose lesions persisted or progressed to CIN2/CIN3, showed AgNOR areas greater than 3.0 µ(2) in 50/55 cases (specificity: 100%; sensitivity: 91%; PPV: 100%; NPV: 80%). CONCLUSIONS: p16INK4a is expressed in a high percentage of returning lesions. AgNOR might be a better marker of proliferation of CIN1 than p16INK4a (PPV = 100%), which means that a value greater than 3.0 µ(2) indicates the persistence or progression of the lesion. As its NPV is 80%, a value of AgNOR area less than 3.0 µ(2) in CIN1 leaves a margin of doubt about the future behavior of the lesion.