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PubMed1999

Gene by environment interaction and asthma.

The term interaction has two specific meanings in epidemiology. The first definition is purely a statistical one. Interaction is defined as the coefficient of the product term of two or more risk factors. An alternative definition is a more biologically oriented one in which interaction between two risk factors is defined as their coparticipation in the same causal mechanism of disease development. There are at least five different types of biological gene by environment interaction. It is important to recognize that clinical or epidemiological studies alone are unable to distinguish between these five different types of biological interaction. Asthma is a prototypical complex trait in which gene by environment interaction is important in disease causation. Environmental factors important in asthma include cigarette smoking (active/passive), allergen exposure, viral and bacterial respiratory illness, occupation, and possibly diet. There are two primary study designs that have been utilized for the assessment of gene by environment interaction: a traditional case control study or a nested case control study or a case control study nested within the larger longitudinal cohort. There are three important methodological issues in the assessment of gene by environment interaction. The first is the issue of misspecification of either genotype or environmental exposure or, for that matter, disease status. The second is possible confounding, and the third is the confounding of interaction with dose response. The four most critical factors for determining sample size for gene by environment interaction studies are the odds ratio for the interaction, the prevalence for exposure in the population, the prevalence of the genotype in the population, and the case to control ratio. In general, sample sizes have been too small to detect meaningful gene by environment interactions.

Animals↗
PubMed2006

The chemical nature of the (plus sign in circle)C-H...X- (X=Cl or Br) interaction in imidazolium halide ionic liquids.

The C-H...X (X=Cl or Br) interaction is traditionally characterized as a relatively weak interaction. However, this interaction becomes very strong in the imidazolium-based halide ionic liquids [J. Phys. Chem. 123, 174501 (2005)]. This strong interaction had been attributed to the electrostatic interaction between the imidazolium cation and the halide anion. In this paper, the chemical nature of the (plus sign in circle)C-H...Cl(-) and ( plus sign in circle)C-H...Br(-) interactions is investigated by atoms in molecules (AIM) and natural bond orbital (NBO) analyses. The AIM calculations indicate that in the EmimX complexes, the (plus sign in circle)C-H...Cl(-) and (plus sign in circle)C-H...Br(-) interactions have some covalent character, especially the (plus sign in circle)C-H...Cl(-) interaction. Mulliken, ChelpG charge, and natural bond orbital population analyses for these two kinds of interactions indicate that the charge transfer is important in the interaction of the cation with the anion. In addition, the NBO analysis demonstrated that the stabilization energy is due to an n-->sigma(C-H) (*) orbital interaction. However, in the Emim2X and Emim3X complexes, the calculated results suggested a dominant electrostatic character for the (plus sign in circle)C-H...Cl(-) and (plus sign in circle)C-H...Br(-) interactions.

Journal Article↗
PubMed1997

Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system.

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.

Insulin-Like Growth Factor I↗
PubMed1996

Intermediate filament protein domain interactions as revealed by two-hybrid screens.

All intermediate filament proteins possess three distinct domains: heads, rod and tail, and subdomains within the rod called helices 1A, 1B, 2A, and 2B. Subunit packing within a filament is a consequence of interactions among these domains. Several such interactions are known, but probably many more contribute to stabilizing filament structure. We examined a number of such potential interactions using the yeast two-hybrid system. Domains or subdomains of murine vimentin, a Type III intermediate filament protein, were fused with either the DNA-binding or trans-activating domain of GAL4, a transcription factor. Interaction between the vimentin domains/subdomains functionally reconstituted GAL4, thereby activating transcription of a GAL1-LacZ reporter gene. The oligomeric state at which the interactions took place, i.e. whether the domains/subdomains were dimeric or tetrameric as they interacted, was also determined. These studies revealed a number of interesting interactions, among which was a strong homotypic binding to helix 2B to form tetramers. They also demonstrated a lack of interaction among others expected to do so based on current structural models. From these results we deduced which of the candidates for interactions, suggested by current models, were true protein-protein interactions and which represented nearest-neighbors only. Thus, the A11 and A22 modes of molecular alignment identified by Steinert et al. (Steinert, P. M., Marekov, L. N., Fraser, R. D. B., and Parry, D. A. D. (1993) J. Mol. Biol. 230, 436-452) are probably true interactions, whereas the A12 and ACN modes may describe adjacent but non-interacting molecules.

Animals↗
PubMed1998

Grb10 interacts differentially with the insulin receptor, insulin-like growth factor I receptor, and epidermal growth factor receptor via the Grb10 Src homology 2 (SH2) domain and a second novel domain located between the pleckstrin homology and SH2 domains.

The Grb10 protein appears to be an adapter protein of unknown function that has been implicated in insulin receptor (IR) signaling. The interaction of this protein with the IR has been shown to be mediated in part by the Src homology 2 (SH2) domain of Grb10. Here we demonstrate the existence of a second novel domain within Grb10 that interacts with the IR and insulin-like growth factor receptor in a kinase-dependent manner. This domain was localized to a region of approximately 50 amino acids, and we term it the BPS domain to denote its location between the PH and SH2 domains. The BPS domain does not bear any obvious resemblance to other known protein interaction domains but is highly conserved among the Grb10-related proteins Grb7 and Grb14. We show that the BPS domain interaction is dependent upon receptor tyrosine kinase activity. Furthermore, interaction of the BPS domain requires the kinase domain of the IR, since mutation of the paired tyrosine residues (Y1150F/Y1151F) within the IR activation loop dramatically reduced the interaction. Last, our data suggest that the presence of two distinct protein interaction domains may help to determine the specificity by which Grb10 interacts with different receptors. Specifically, the IR, which appears to interact most strongly with Grb10, interacts well with both the SH2 and BPS domains. Conversely, the insulin-like growth factor receptor and EGFR, which interact less avidly with Grb10, interact well only with the BPS domain or the SH2 domain, respectively. In summary, our findings demonstrate the existence of a previously unidentified tyrosine kinase activity-dependent binding domain located between the Pleckstrin homology and SH2 domains of Grb10.

Amino Acid Sequence↗
PubMed1998

The corepressor N-CoR and its variants RIP13a and RIP13Delta1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70.

Repression of transcription by the classical nuclear receptors (e.g. TR, RAR), the orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mxi-1 and Mad bHLH-zip proteins and the oncoproteins PLZF and LAZ3/BCL6 is mediated by the corepressors N-CoR and SMRT. The interaction of the corepressors with the components involved in chromatin remodelling, such as the recruiting proteins Sin3A/B and the histone deacteylases HDAc-1 and RPD3, has been analysed in detail. The N-CoR/Sin3/HDAc complexes have a key role in the regulation of cellular proliferation and differentiation. However, the interaction of these corepressors with the basal transcriptional machinery has remained obscure. In this study we demonstrated that the N-terminalrepression domains and the receptor interactiondomains (RID) of N-CoR and its splice variants, RIP13a and RIP13Delta1, directly interact with TAFII32 in vivo and in vitro . We show that interaction domain II within the N-CoR and RIP13a RID is required for the interaction with TAFII32. We also observed that N-CoR directly interacts with each of the basal factors, TFIIB and TAFII70, and can simultaneously interact with all three basal factors in a non-competitive manner. Furthermore, we provide evidence that suggests the RVR/Rev-erbbeta-corepressor complex also interacts with the general transcriptional machinery, and that the physicalassociation of TFIIB with N-CoR also occurs in the presence of Sin3B and HDAc-1. Interestingly, we observed that N-CoR expression ablated the functional interaction between TFIIB and TAFII32 that is critical to the initiation of transcription. In conclusion, this study demonstrates that the N-terminal repressor region and the C-terminal RIDs are part of the corepressor contact interface that mediates the interaction with the general transcription factors, and demonstrates that TAFs can also directly interact with corepressors to mediate signals from repressors to the basal machinery. We also suggest that N-CoR interacts with the central components of the transcriptional initiation process (TFIIB, TAFs) and locks them into a non-functional complex or conformation that is not conducive to transcription.

Animals↗
PubMed2001

The yeast protein interaction network evolves rapidly and contains few redundant duplicate genes.

In this paper, the structure and evolution of the protein interaction network of the yeast Saccharomyces cerevisiae is analyzed. The network is viewed as a graph whose nodes correspond to proteins. Two proteins are connected by an edge if they interact. The network resembles a random graph in that it consists of many small subnets (groups of proteins that interact with each other but do not interact with any other protein) and one large connected subnet comprising more than half of all interacting proteins. The number of interactions per protein appears to follow a power law distribution. Within approximately 200 Myr after a duplication, the products of duplicate genes become almost equally likely to (1) have common protein interaction partners and (2) be part of the same subnetwork as two proteins chosen at random from within the network. This indicates that the persistence of redundant interaction partners is the exception rather than the rule. After gene duplication, the likelihood that an interaction gets lost exceeds 2.2 x 10(-3)/Myr. New interactions are estimated to evolve at a rate that is approximately three orders of magnitude smaller. Every 300 Myr, as many as half of all interactions may be replaced by new interactions.

Evolution, Molecular↗
PubMed1998

Yeast two-hybrid: so many interactions, (in) so little time...

Protein-protein interactions are essential to cellular mechanisms at all levels in biologically responsive systems. These interactions occur extracellularly and include ligand-receptor interactions, cell adhesion, antigen recognition, and virus-host recognition. Intracellular protein-protein interactions occur in the formation of multi-protein complexes, during the assembly of cytoskeletal elements, and between receptor-effector, as well as effector-effector, molecules of signal transduction pathways. Finally, assembly of transcriptional machinery involves protein interactions. The yeast two-hybrid method is a powerful technique for analyzing these protein-protein interactions. Since the publication of this technique in the late 1980s, the robust nature and far-reaching utility of yeast two-hybrid systems for functional expression library cloning has led to the identification of many novel proteins in all areas of biological life science research. Additionally, two-hybrid techniques provide a rapid and versatile system for the further characterization of discrete protein-protein interactions. Recent variations on the basic system have enabled application well beyond protein pairs, to investigate multi-protein complexes and protein-nucleotide interactions. Yeast two-hybrid methods necessitate expression and subsequent interaction between a "protein of interest" functional pair within the yeast cell, ultimately driving reporter gene expression and thus effectively linking protein-protein interaction(s) to a change in yeast cell phenotype. Functional protein-protein interactions using the two-hybrid techniques have been demonstrated for all levels of cellular biology; however, until recently, extracellular protein-protein interactions were excluded from investigations using this technique. Investigations from several labs have now demonstrated that extracellular proteins can be studied using two-hybrid methods, thereby enabling intense study of extracellular protein partners using the robust nature and the genetic power of yeast.

Animals↗
PubMed2004

Weak interactions, omnivory and emergent food-web properties.

Empirical studies have shown that, in real ecosystems, species-interaction strengths are generally skewed in their distribution towards weak interactions. Some theoretical work also suggests that weak interactions, especially in omnivorous links, are important for the local stability of a community at equilibrium. However, the majority of theoretical studies use uniform distributions of interaction strengths to generate artificial communities for study. We investigate the effects of the underlying interaction-strength distribution upon the return time, permanence and feasibility of simple Lotka-Volterra equilibrium communities. We show that a skew towards weak interactions promotes local and global stability only when omnivory is present. It is found that skewed interaction strengths are an emergent property of stable omnivorous communities, and that this skew towards weak interactions creates a dynamic constraint maintaining omnivory. Omnivory is more likely to occur when omnivorous interactions are skewed towards weak interactions. However, a skew towards weak interactions increases the return time to equilibrium, delays the recovery of ecosystems and hence decreases the stability of a community. When no skew is imposed, the set of stable omnivorous communities shows an emergent distribution of skewed interaction strengths. Our results apply to both local and global concepts of stability and are robust to the definition of a feasible community. These results are discussed in the light of empirical data and other theoretical studies, in conjunction with their broader implications for community assembly.

Computer Simulation↗
PubMed2001

Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1-E2 interaction.

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1-E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1-311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315-605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N-E2TAD interaction is not essential for replication function and is probably involved in some other E1-E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

Animals↗
PubMed2007

Three functional subdomains of the Escherichia coli FtsQ protein are involved in its interaction with the other division proteins.

FtsQ, an essential protein for the Escherichia coli divisome assembly, is able to interact with various division proteins, namely FtsI, FtsL, FtsN, FtsB and FtsW. In this paper, the FtsQ domains involved in these interactions were identified by two-hybrid assays and co-immunoprecipitations. Progressive deletions of the ftsQ gene suggested that the FtsQ self-interaction and its interactions with the other proteins are localized in three periplasmic subdomains: (i) residues 50-135 constitute one of the sites involved in FtsQ, FtsI and FtsN interaction, and this site is also responsible for FtsW interaction; (ii) the FtsB interaction is localized between residues 136 and 202; and (iii) the FtsL interaction is localized at the very C-terminal extremity. In this third region, the interaction site for FtsK and also the second site for FtsQ, FtsI, FtsN interactions are located. As far as FtsW is concerned, this protein interacts with the fragment of the FtsQ periplasmic domain that spans residues 67-75. In addition, two protein subdomains, one constituted by residues 1-135 and the other from 136 to the end, are both able to complement an ftsQ null mutant. Finally, the unexpected finding that an E. coli ftsQ null mutant can be complemented, at least transiently, by the Streptococcus pneumoniae divIB/ftsQ gene product suggests a new strategy for investigating the biological significance of protein-protein interactions.

Bacterial Proteins↗
PubMed2005

Soliton interaction in a fiber ring laser.

We have experimentally investigated the soliton interaction in a passively mode-locked fiber ring laser and revealed the existence of three types of strong soliton interaction: a global type of soliton interaction caused by the existence of unstable cw components, a local type of soliton interaction mediated through the radiative dispersive waves, and the direct soliton interaction. We found that the appearance of the various soliton operation modes observed in the passively mode-locked fiber soliton lasers are the direct consequences of these three types of soliton interactions. The soliton interaction in the laser is further numerically simulated based on a pulse tracing technique. The numerical simulations confirmed the existence of the dispersive-wave-mediated soliton interaction and the direct soliton interaction. Furthermore, it was shown that the resonant dispersive-wave-mediated soliton interaction in the laser always has the consequence of causing random irregular relative soliton movement and the experimentally observed states of bound solitons are caused by the direct soliton interaction. In particular, as the solitons generated in the laser could have a profile with long tails, the direct soliton interaction could extend to a soliton separation that is larger than 5 times the soliton pulse width.

Journal Article↗
PubMedDate not supplied

Dynamic interactions in physically realistic collaborative virtual environments.

This work describes our efforts in creating a general object interaction framework for dynamic collaborative virtual environments. Furthermore, we increase the realism of the interactive world by using a rigid body simulator to calculate all actor and object movements. The main idea behind our interactive platform is to construct a virtual world using only objects that contain their own interaction information. As a result, the object interactions are application independent and only a single scheme is required to handle all interactions in the virtual world. In order to have more dynamic interactions, we also created a new and efficient way for human users to dynamically interact within virtual worlds through their avatar. In particular, we show how inverse kinematics can be used to increase the interaction possibilities and realism in collaborative virtual environments. This results in a higher feeling of presence for connected users and allows for easy, on-the-fly creation of new interactions. For the distribution of both the interactive objects and the dynamic avatar interactions, we keep the network load as low as possible. To demonstrate the effectiveness of our techniques, we incorporate them into an existing CVE framework.

Computer Simulation↗
PubMed1979

Studies on protein folding, unfolding and fluctuations by computer simulation. III. Effect of short-range interactions.

The theoretical model of proteins on the two-dimensional square lattice, introduced previously, is extended to include the specific short-range interactions. Attractive long-range interactions with various specificities and non-specific repulsive long-range interactions in the form of self-avoidance of the polymer chain are also operative in the model. Dynamics of the model protein is studied by a Monte Carlo method. The short-range interactions are found to accelerate the folding and unfolding transitions. Non-specific part of the attractive long-range interactions have a competing effect of decelerating the transitions. When the short-range interactions are weighted beyond a certain extent over the attractive long-range interactions are weighted beyond a certain extent over the attractive long-range interactions, the all-or-none character of the folding and unfolding transitions is destroyed. How the destruction proceeds is quantitatively expressed in terms of the S-H curves. The limiting case of dominance of the specific short-range interactions over the attractive long-range interactions is studied in detail. The lattice polymer in this limit does not behave like a globular protein at all. This observation leads to a reexamination of the currently popular notion of the dominance of the short-range interactions. A new concept of consistency is proposed to replace it. Possible mechanisms of the acceleration of the transitions by the specific short-range interactions are discussed.

Computers↗
PubMed1991

Horizontal interactions between visual cortical neurones studied by cross-correlation analysis in the cat.

1. To explore the functional significance of horizontal neural connections in the extent of a 'hypercolumn' of the cat visual cortex, we carried out cross-correlation analysis of spike trains recorded simultaneously from a pair of neurones separated horizontally by less than 1 mm. 2. Significantly correlated firings, which were found in sixty-eight pairs of cells among 327 pairs analysed, were classified into three types on the basis of their functional implications: (1) excitatory interactions, (2) inhibitory interactions and (3) common inputs to both neurones of a pair from other sources. 3. Of these three types, common inputs were encountered most frequently. Excitatory interactions were always accompanied by common inputs. Inhibitory interactions were observed least frequently. 4. The proportion of cell pairs with correlated firings was high in pairs with a horizontal separation of less than 200 microns and decreased markedly with a horizontal separation of more than 400 microns. 5. Regarding laminar locations of cells, common inputs and excitatory interactions were often observed in layers II + III and V, whereas laminar bias was not seen in inhibitory interactions. 6. With respect to difference in orientation preference between two cells, all the three types of correlations were observed, mostly in cell pairs with a difference of less than 45 deg. In particular, common inputs and excitatory interactions were often seen in cell pairs with matched orientation preferences, but inhibitory interactions were found mostly in those with slightly different orientation preferences. In addition, common inputs and excitatory interactions tended to be found between cells with the same eye preference. 7. These results suggest that horizontal functional interactions exist mainly in a range of up to 400 microns as far as the extent of a hypercolumn of the visual cortex is concerned, and these interactions operate effectively between cortical cells with similar receptive field properties except for inhibitory interactions.

Action Potentials↗
PubMed1996

Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein.

Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.

Amino Acid Sequence↗
PubMed2000

Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells.

Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.

Animals↗
PubMed2003

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells.

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.

Amino Acid Sequence↗