Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the periGolgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFPRab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFPRab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.Chlamydiae are major bacterial pathogens of ocular, urogenital, and pulmonary mucosal surfaces (51). Infections caused by Chlamydia trachomatis are the leading cause of bacterially acquired sexually transmitted disease (10), as well as of preventable blindness worldwide (64). In addition, Chlamydia pneumoniae infections are major causes of upper respiratory tract infections and have recently been linked to chronic heart disease (24, 25). Chlamydiae are obligate intracellular bacteria that replicate within a nonacidified vacuole termed an inclusion (26). Within the inclusion, chlamydiae undergo a biphasic developmental cycle that alternates between the infectious metabolically inactive elementary body (EB) and the noninfectious metabolically active reticulate body (40). Although chlamydiae enter nonprofessional phagocytes by multiple mechanisms (reviewed in reference 26), once the chlamydiae are internalized, they actively modify the properties of the nascent vacuole during the first 2 h postinfection, resulting in trafficking of the inclusion to the peri-Golgi region, fusion of the inclusion with a subset of Golgi-derived exocytic vesicles, and avoi...

Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Gal␤1,4Man␣1-PO 4 ) repeating units. In L. major these are extensively substituted with Gal(␤1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain ␤1,3-galactosyltransferases (␤GalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain ␤GalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression.The trypanosomatid protozoan parasite Leishmania infects over 12 million people worldwide, causing a variety of diseases that range from mild cutaneous lesions to fatal visceral infections (1). Within vertebrates Leishmania resides within acidified phagosomes of macrophages as the amastigote stage. A key step of the infectious cycle is the ability of the parasite to be transmitted to fresh hosts by an insect vector, phlebotomine sand flies. Several studies have emphasized the importance of lipophosphoglycan (LPG), 1 an abundant surface glycolipid of Leishmania promastigotes, in sand fly survival (reviewed in Refs. 2-4). Following a sand fly bite, Leishmania and the blood meal are enclosed by a midgut peritrophic matrix for several days, whereas parasites differentiate to the replicating procyclic promastigote stage. During this period LPG and other phosphoglycans (PGs) contribute to survival in the hydrolytic milieu of the midgut (3). After a few days the matrix is degraded and the remnants of the blood meal are excreted; at this time, promastigotes bind to midgut epithelium through an LPG-dependent interaction to avoid being excreted as well (5). As digestion is completed and the fly prepares to feed again, parasites differentiate to the infectious metacyclic stage, which synthesize a structurally modified metacyclic form LPG that is unable to bind the midgut (5-7). The detached metacyclic parasites are adapted for transmission and establishment of the infection in a new vertebrate host (8).The basic "backbone" structure in all Leishmania consists of a 1-O-alkyl-2-lyso-phosphatidylinositol lipid anchor and heptasaccharide core, to which is joined a long PG polymer composed of 15-30 (Gal␤1,4Man␣1-PO 4 ) repeating units, terminated by a capping oligosaccharide (Fig. 1). In different species and/or developmen...

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Studies to localize the herpes simplex virus 1 portal protein encoded by U L 6, the putative terminase components encoded by U L 15, U L 28, and U L 33, the minor capsid proteins encoded by U L 17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pU L 6, pU L 15, pU L 17, pU L 28, or pU L 33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pU L 6, pU L 17, pU L 28, and pU L 33. Epitopes of pU L 15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pU L 6, pU L 17, pU L 28, pU L 33, and at least some portion of pU L 15 are located at the external surface of the capsid.Capsids form in the nuclei of cells infected with all herpesviruses. Herpes simplex virus (HSV) capsid pentons and hexons form spontaneously from five and six molecules of ICP5, respectively; these capsomeres are linked by triplexes consisting of two molecules of VP23 and one molecule of VP19C to form a porous procapsid (23,36,43). ICP5 is also associated with ICP35, which forms an internal shell or scaffold within the procapsid. The procapsid is believed to give rise to the three other types of capsids seen in HSV-infected cells, designated types A, B, and C. All of these capsids differ internally but contain identical outer shells, as determined by cryoelectron microscopy (21,35,49). Type B capsids retain the scaffold internal to the outer shell, type A capsids contain only the outer shell, and type C capsids lack the internal scaffold but contain viral DNA (14). Type C capsids then bud from the nuclear membrane in a reaction termed primary envelopment (19,32).One of the vertices of A, B, and C capsids is biochemically and structurally unique and has been designated the portal vertex. Thus, the U L 6-encoded protein (pU L 6) forms a dodecameric ring with an internal diameter of at least 65 Å, i.e., sufficiently wide to accommodate DNA as it is packaged into the capsid (44). Critical to the discovery of the portal was the observation that an antibody to the C terminus of pU L 6 recognized epitopes on a single vertex of type B capsids, thus showing that at least the C terminus of pU L 6 is located at the capsid exterior in a position to access incoming viral DNA (22, 39).It has also been shown that HSV-1 B capsids contain a number of capsid proteins in addition to triplexes, pU L 6, ICP5, and ICP35. These proteins include approximately 1.2 copies of pU L 15, 2.4 copies of pU L 28, 27 to 42 copies of pU L 25, 19.2 copies of pU L 17, and an undetermined amount of pU L 33 (6,7,15,25,26,33,41,42,48). By analogy to extensive studies of bacteriophage capsid assembly, it might be predicted that some of these minor capsid proteins would be involved in processing concatameric DNA and threading the DNA into the portal through the hydrolysis of ATP (9). Such a complex, termed the terminase, remains somewhat enigmati...

Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of icosahedral herpes simplex virus capsids. pUL17 and pUL25, which form the capsid vertex-specific component (CVSC), particularly contributed to capsid resilience along the 5-fold and 2-fold but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry of the protein composition of the capsids, revealed that both pUL17 and pUL25 are required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids. In this study, the structural, material properties of herpes simplex virus 1 were investigated. The capsid of herpes simplex virus is built up of a variety of proteins, and we scrutinized the influence of two of these proteins on the stability of the capsid. For this, we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach, we revealed that both studied proteins play an essential role in viral stability. These new insights support us in forming a complete view on viral structure and furthermore could possibly help not only to develop specific antivirals but also to build protein shells with improved stability for drug delivery purposes.

Herpesvirus virions are composed of a double-stranded DNA genome encapsidated in an icosahedral shell, an amorphous proteinaceous network surrounding the capsid termed the tegument, and a glycoprotein-decorated envelope surrounding the tegument (reviewed in references 25 and 35). The predominant model of virion assembly involves primary envelopment of the nucleocapsid at the inner nuclear membrane (INM), fusion of this nascent virion envelope with the outer nuclear membrane (ONM), and subsequent attachment of tegument proteins to the de-enveloped nucleocapsid in a region of the cytoplasm derived from the Golgi apparatus and/or transGolgi network (25,34). The fact that the bulk of the tegument is applied at a step after primary envelopment is consistent with the relatively sparse electron microscopic appearance of the perinuclear virion tegument as opposed to the denser tegument of the extracellular virion (3,15). This model of virion egress suggests opportunities for a subset of tegument proteins to attach directly or indirectly to the nucleocapsid in either the nucleosol or cytosol or during budding into nuclear or cytoplasmic membranes. Supporting the idea that at least some tegumentation occurs in the nucleoplasm are the observations that pU L 36, pU L 37, and vhs (the U L 41 gene product) are associated with intranuclear capsids (5, 30). Budding through the INM likely causes incorporation of another set of proteins into the tegument, including the peripheral membrane proteins pU L 11 and pU L 31, the viral kinase encoded by U S 3, and the nucleoplasmic proteins VP16 and VP22 (encoded by U L 48 and U L 49, respectively) (2, 27, 29, 31). Of these, only the pU L 31 gene product is absent from extracellular virions, indicating its loss at the de-envelopment step (13,22,31).

Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23,29,47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U L 6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U L 18 gene product, VP23, and one copy of the U L 38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U L 26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11,12,19,25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U L 15, U L 17, U L 28, U L 32, and U L 33 genes (2,4,10,18,26,28,35,46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U L 25 and U L 17 open reading frames (1,17,20). These proteins are located on the external surface of the viral capsid (24,36,44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU L 25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U L 17 and U L 25 proteins (pU L 17 and pU L 25, respectively) associate with all capsid types, and pU L 17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28,36,37). How the U L 17 and U L 25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU L 17 and pU L 25 become incorporated into the capsid during the assembly pathway. Less pU L 25 associates with pU L 17(Ϫ) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU L 25 associates with capsids lacking DNA (30,36).Both pU L 25 and pU L 17 are necessary for proper nucleocapsid assembly, but their respective de...

ZusammenfassungThrombosen der Koronararterien bilden sich bevorzugt in geometrisch komplizierten Gefäßabschnitten. Hier entstehen Sekundärströmungen mit Geschwindigkeitskomponenten senkrecht gegen die Wand, oder sogar Staupunktströmungen, in denen Stromlinien senkrecht gegen die Wand gerichtet sind. Strömungsexperimente an Glasmodellen solcher Gefäßabschnitte ergaben, daß Blutpartikel (Thrombozyten, Erythrozyten) insbesondere durch Staupunktströmungen gegen die Wand transportiert und mit ihr in Berührung gebracht werden. Bei langsamen Staupunktströmungen bilden die Partikel im Staugebiet wandständige Mikro-thromben, sofern ihre Adhäsivität groß genug ist, um der Schubkraft der entlang der Wand abfließenden Strömung zu widerstehen. Die von schnellen Staupunktströmungen erzeugten hohen Wandschubkräfte wirken der Ablagerung entgegen. Wegen der geometrischen Übereinstimmung der Glasmodelle mit bevorzugt von Thrombosen befallenen Abschnitten der Koronararterien und wegen der Übereinstimmung der Ablagerungsor-te innerhalb der Lichtung von Blutgefäß und dessen Modell war zu vermuten, daß dieser Zusammenhang auch für die Thrombogenese in vivo gilt.Zur Überprüfung dieser Hypothesen wurden die strömungsmechanischen Bedingungen für die Entstehung einer Plättchenmikrothrombose durch eine rotationssymmetrische Staupunktströmung in vitro simuliert. Als Staufläche diente eine Glaswand, als Fluid strömte plättchenreiches Plasma von Patienten, bei denen das Thromboserisiko und die Wirkung von Hämostatika festgestellt werden sollte. Während der Strömung wurde das Wachstum der auf der Glaswand entstehenden Thrombozyten- »Mikro-thromben« fortlaufend registriert.Diese Anordnung reduziert die intravitalen Verhältnisse auf ein Minimalsystem bestehend aus drei Komponenten, welche bei der Entstehung der wandständigen Thrombose in vivo unumgänglich beteiligt sind: Blut, Strömung, Wand. Sie bildet also als Minimalsystem einen pathophysiolo-gischen Vorgang im Modell nach. Bei Patienten mit klinischen Anzeichen erhöhter Gerinnungsneigung. (z.B. Herzinfarkt, 3 Tage nach der Messung) entstanden auch in vitro »Mi-krothrombosen«. Anhand der Ergebnisse wird diskutiert, ob sich diese Staupunktanordnung, die den patho-physiologischen Vorgang der Thrombogenese offensichtlich im Modell nachzubilden gestattet, auch dazu eignet, die individuelle Adhäsions- und Aggregationsneigung der Thrombozyten zu messen.