Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^

High-resolution multi-proxy record of the last glacial period from Lake Van, eastern Anatolian high plateau, Turkey

Detailed analyses of the Lake Van pollen, Ca/K ratio and stable oxygen isotope record allow the identification of millennial-scale vegetation and environmental changes in eastern Anatolia throughout the last glacial (~75-15 ka BP). The climate within the last glacial was cold and dry, with low arboreal pollen (AP) levels. The driest and coldest period corresponds to Marine Isotope Stage (MIS) 2 (~28-14.5 ka BP) dominated by the highest values of xerophytic steppe vegetation. Our high-resolution multi proxy record shows rapid expansions and contractions of tree populations that reflects variability in temperature and moisture availability. This rapid vegetation and environmental changes can be linked to the stadial-interstadial pattern of the Dansgaard-Oeschger (DO) events as recorded in the Greenland ice cores. Periods of reduced moisture availability were characterized by enhanced xerophytic species and high terrigenous input from the Lake Van catchment area. Furthermore, comparison with the marine realm reveals that the complex atmosphere-ocean interaction can be explained by the strength and position of the westerlies, which is responsible for the supply of humidity in eastern Anatolia. Influenced by diverse topography of the Lake Van catchment, larger DO interstadials (e.g. DO 19, 17-16, 14, 12 and 8) show the highest expansion of temperate species within the last glacial. However, Heinrich events (HE), characterized by highest concentrations of ice-rafted debris (IRD) in marine sediments, are identified in eastern Anatolia by AP values not lower and high steppe components not more abundant than during DO stadials. In addition, this work is a first attempt to establish a continuous microscopic charcoal record over the last glacial in the Near East, which documents an initial immediate response to millennial-scale climate and environmental variability and enables us to shed light on the history of fire activity during the last glacial.

High-resolution multi-proxy record of the last interglacial period from Lake Van, eastern Anatolian high plateau, Turkey

A high-resolution multi-proxy record from Lake Van, eastern Anatolia, derived from a lacustrine sequence cored at the 357 m deep Ahlat Ridge (AR), allows a comprehensive view of paleoclimate and environmental history in the continental Near East during the last interglacial (LI). We combined paleovegetation (pollen), stable oxygen isotope (d18Obulk) and XRF data from the same sedimentary sequence, showing distinct variations during the period from 135 to 110 ka ago leading into and out of full interglacial conditions. The last interglacial plateau, as defined by the presence of thermophilous steppe-forest communities, lasted ca. 13.5 ka, from ~129.1-115.6 ka BP. The detailed palynological sequence at Lake Van documents a vegetation succession with several climatic phases: (I) the Pistacia zone (ca. 131.2-129.1 ka BP) indicates summer dryness and mild winter conditions during the initial warming, (II) the Quercus-Ulmus zone (ca. 129.1-127.2 ka BP) occurred during warm and humid climate conditions with enhanced evaporation, (III) the Carpinus zone (ca. 127.2-124.1 ka BP) suggest increasingly cooler and wetter conditions, and (IV) the expansion of Pinus at ~124.1 ka BP marks the onset of a colder/drier environment that extended into the interval of global ice growth. Pollen data suggest migration of thermophilous trees from refugial areas at the beginning of the last interglacial. Analogous to the current interglacial, the migration documents a time lag between the onset of climatic amelioration and the establishment of an oak steppe-forest, spanning 2.1 ka. Hence, the major difference between the last interglacial compared to the current interglacial (Holocene) is the abundance of Pinus as well as the decrease of deciduous broad-leaved trees, indicating higher continentality during the last interglacial. Finally, our results demonstrate intra-interglacial variability in the low mid-latitudes and suggest a close connection with the high-frequency climate variability recorded in Greenland ice cores.

Paleoclimatate of the Middle Burdigalian-Langhian period in Turkey

The Miocene is the last warm episode in Earth history, and this episode was well recorded in Turkey as shown by plant distribution and inferred numerical temperature values. In this study, Ören-Kultak, Hüssamlar and Karacaagac palynofloras from western Turkey, which are characterized by the thermophilous plants (Engelhardia, Sapotaceae, Cyrillaceae, Avicennia, Arecaceae, Palmae), are described. Age determinations of these palynofloras (middle Burdigalian-Langhian) are strengthened by the mammalian fossil record (MN4-5) and strontium isotope results. Palaeoclimate is humid and warm subtropical during the middle Burdigalian-Langhian time interval in Europe and Turkey. However, temperature difference has been observed between Europe and Turkey during this time interval and it could be explained by the palaeogeographic position of countries. Despite some discrepancies in the climatic values and palaeovegetation groups, warm climatic conditions are recorded, based on the palynofloras, in Turkey (Cayyrhan, Havza, Can, Etili, Gönen, Bigadic, Emet, Kirka and Kestelek, Sabuncubeli, Soma, Tire, Kulogullary, Bascayyr, Hüssamlar and Karacaagac), Greece and elsewhere in Europe throughout the middle Burdigalian-Langhian period. This warming is related to the Middle Miocene Climatic Optimum period. Carbon and oxygen isotope values obtained from tooth enamel of Gomphotherium sp. from Kultak and Hüssamlar indicate similar ecological condition during the Burdigalian-Langhian time. This isotopic result and high MAPDRY value from the Kultak locality are in agreement with ecological interpretation of mammalian fossils. Besides, according to the precipitation values, central and northwestern Anatolian sites provide more rainfall during the Burdigalian-Langhian time interval than the western Anatolian sites.

Neogene palaeoclimate reconstructions in Anatolia (Turkey)

Neogene basins are widespread in Turkey and contain important lignite deposits. In this study, we reconstruct quantitatively the Late Oligocene-Miocene climate evolution in western and central Anatolia by applying the Coexistence Approach to the palynoflora. The obtained results are compared with the data derived from the published and ongoing studies in western and central Anatolia palynofloras by application of the Coexistence Approach. The Coexistence Approach results show that the sedimentation mainly developed on terrestrial environment under the warm subtropical climatic conditions and marine influence during the Chattian and Aquitanian period in western Anatolia (16.5-21.3°C of mean annual temperature (MAT) and 5.5-13.3°C of mean temperature of coldest month (CMT)). After the regression of the sea during the Burdigalian period, the vegetation developed under the terrestrial conditions, which had started in the Burdigalian time in western and central Anatolia and continued in the early-middle Serravallian period. Warm subtropical climate is suggested during the Chattian and Aquitanian period in western Anatolia and becomes cooler in subtropical conditions because of decreasing of the P/A-ratio during the latest Burdigalian-Langhian. The climate was subtropical in western and central Anatolia during the Early-Late Serravalian due to the increasing of the subtropical elements (17.2 to 20.8°C of MAT and 9.6 to13.1°C of CMT). Besides, decreasing of the CMT and MAT values in western and central Anatolia supports the latest Chattian-earliest Aquitanian warming and middle Miocene climatic optimum that is also globally observed. Warm temperate climatic conditions are observed in the Late Miocene. During the early-middle Tortonian, the values are 15.6 to 20.8°C for the MAT, 5.5 to 13.3°C for the CMT and 823 and 1520 mm for the mean annual precipitation (MAP). They had also dry seasons due to lower boundary of MAP lying at 823mm during the middle-Late Tortonian. The palaeotopography of central Anatolia was higher when compared to that of western Anatolia because dominance of the mountain forests was present during the Middle-Late Miocene in central Anatolia. This study provides the first quantitative model for Late Oligocene-Miocene palaeoclimatic evolution in western and central Anatolia.

(Table 2) Paleoclimate reconstruction for palynology samples of Turkey

Palynofloras of the Kocaçay and Cumaovasi basins in western Turkey that belong to a time-span from the late Early to late Middle Miocene (the late Burdigalian-Serravallian) are studied and compared with published palynofloras of Europe and Turkey. Palynological data and numerical climatic results obtained by the coexistence approach indicate palaeoclimate changed from warm subtropical to temperate during the late Burdigalian-Serravallian. Moreover, the palaeoclimates of the Kocacay and Cumaovasi basins are compared with continental palaeoclimatic records of coal-bearing sediments in western Turkey and current temperatures in the Izmir region. According to this comparison, palaeoclimatic results of these basins and other localities in western Turkey show a distinct difference as a result of orographic change. The palaeovegetation in the Kocaçay and Cumaovasi basins during the studied time-span was affected by palaeotopography and palaeoclimate. In these basins mixed mesophytic, coniferous forests, and swamp palaeovegetation generally predominated during the late Early-early Middle Miocene. The role of the herbaceous taxa increased at the end of the late Middle Miocene (the Serravallian) in the Kocaçay and Cumaovasi basins. It is obvious from the palynomorph data of these basins that grassland palaeovegetation started to expand in the late Middle Miocene. Unlike in Central Europe, where late Burdigalian and Langhian represent a period of outstanding warmth, the so-called Mid-Miocene Climatic Optimum, cold month mean temperatures reconstructed in this study point to an ongoing cooling trend, already from the late Burdigalian onwards, possibly related to increasing terrestrial conditions in the study area.