This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.SUPPORT: NIH 5R01AI052341-02 'Nucleocapsid envelopment Herpes simplex virus-1', 03/03-02/08Joel D. Baines Cornell UniversityNIH 2R01GM050740-09 'Herpes Simplex Virus Terminases', 01/95-12/07Joel D. Baines Cornell UniversityNIH F32GM067519 'ATP hydrolysis in HSV DNA packaging' to C.L. Duffy, 2003-2005.C.L. Duffy Cornell UniversityABSTRACT:Extracellular herpesvirus virions consist of the nucleocapsid surrounded by an amorphous proteinaceous layer called the tegument that is in turn surrounded by a lipid viral envelope. The latter is derived from host membranes from which virions bud. The particles are known to bud from the inner nuclear membrane where they acquire an initial virion envelope. Enveloped virions then accumulate between the leaflets of the nuclear membrane, and the virion envelope eventually fuses with the outer nuclear membrane leaflet, dumping the de-enveloped nucleocapsid into the cytoplasm, where it eventually becomes re-enveloped and is secreted into the extracellular space. The involvement of possible host cytoskeletal elements in the production of virions can only be studied by the use of electron tomography because tomography allows analysis of individual particles in-situ and without bias due to symmetry-averaging. Preliminary observations have changed the existing paradigm of how the envelopment process occurs, and suggest that herpesviruses exploit the cell in ways that were not predicted. Herpesviruses cause a number of important medical conditions in humans including blindness and encephalitis in both immune-compromised and immune competent patients. The ultimate goal of this study is to understand the mechanisms by which herpesviruses become enveloped at the nuclear membrane and their pathway of egress from the perinuclear space. All undergo the same pathway of virion production and exit, thus the studies will establish the paradigm for this important group of pathogens. The conservation of these steps suggest that it is conceivable that compounds that interfere with this pathway would be effective against all herpesviruses. The characterization of herpesvirus particles using tomography will provide a novel view of the virion egress pathway, and new insight for structure and function of virions and viral proteins.
The first aim i n this project is to elucidate the structure of wild type and mutant particles as they engage the envelopment machinery at the inner nuclear membrane. The nature of the envelopment machinery at the inner nuclear membrane is unknown, although the Baines laboratory has identified three candidates (genes UL11, UL31 and UL34) as important for the envelopment reaction (Baines and Roizman, 1992; Reynolds et al., 2001). Cells infected with wild type and available mutant viruses lacking genes encoding these proteins will be examined by cryoelectron tomography. Preliminary analyses of wild type nascent virions (those located in the perinuclear space), using fixed material, indicate the presence of novel fibers that bridge the space between the herpes simplex virus (HSV) virion envelope and the nucleocapsid. The deduced mass of these fibers is higher than can be accounted for by any viral gene, suggesting the novel possibility that host proteins are used as a structural component during virus assembly. This would be unprecedented. The diameter of the fibers is consistent with cytoskeletal proteins such as actin or lamins. Although the existence of nuclear actin was controversial, its presence is now widely acknowledged, but remains functionally obscure (Bettinger et al., 2004). The fibers are connected to the nucleocapsid in an asymmetrical fashion, and it will be important to verify that this asymmetry is not an artifact of the fixation process, thus cryoelectron microscopy is essential. To test the logical hypotheses that one or more of the envelopment proteins are involved in recruiting the fibers into virions, analysis of the mutant viruses will be performed. If a given protein is involved in recruitment or formation of the fibers, such fibers should be absent or misshapen in particles located at the inner nuclear membrane of cells infected with the relevant mutant virus. A separate, but related issue is whether tegument proteins are incorporated at this stage of the egress pathway. Current dogma suggests that tegument proteins are incorporated only in the cytoplasm after de-envelopment. The structure of individual particles in the perinuclear space will indicate whether at least a subset of tegument proteins are brought into the virion through interaction with the nucleocapsid or the inner nuclear membrane.
The second aim i s to compare the structure of extracellular virions with that of nascent virions. The comparison might indicate that certain steps occur in the egress pathway occur that have only been alluded to in the past. For example, the hypothesis that additional proteins are incorporated along with nucleocapsids as they become enveloped in the cytoplasm is a longstanding one, and predicts the presence of additional mass in the tegument of extracellular virions (Gr newald et al., 2003) as opposed to nascent virions. Our initial tomography work at the RVBC was with plastic sections of conventionally-processed cells. We saw a specialization of the inner leaflet of the nuclear envelope adjacent to, and conforming to, the icosohedral shape of an internal virus particle. We also saw evidence that the nuclear envelope is the origin of viral 'transport vesicles' (manuscript in preparation). However, conventional preparation was inadequate to reliably preserve the finer structural details of the virions and their immediate surroundings. Ultimately, we feel that the 'native' preparation afforded by frozen-hydrated sections will be required. While awaiting establishment of protocols to do this work with frozen-hydrated sections, we are continuing our work by using high-pressure frozen, freeze-substituted material. Our experience with high-pressure freezing will help us establish optimal freezing protocols with our material prior to starting the frozen-hydrated section work.Electron tomography is the only technique that can yield 3-D ultrastructural information on individual virus particles in-situ. In order to be confident of the results, the native preservation of frozen-hydrated preparation will be required. The technique of frozen-hydrated sections, developed at the RVBC, will be a great asset for our work.References1. Baines,J.D. and Roizman,B. (1992). The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells. J. Virol. 66, 5168-5174.2. Bettinger,B.T., Gilbert,D.M., and Amberg,D.C. (2004). Actin up in the nucleus. Nat. Rev. Mol. Cell Biol. 5, 410-415.3. Gr newald,K., Desai,P., Winkler,D.C., Heymann,J.B., Belnap,D.M., Baumeister,W., and Steven,A.C. (2003). Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302, 1396-1398.4. Reynolds,A.E., Ryckman,B., Baines,J.D., Zhou,Y., Liang,L., and Roller,R.J. (2001). UL31 and UL34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J. Virol. 75, 8803-8817.In the previous reporting period, eight new double-tilt tomographic reconstructions were made from the high-pressure frozen material, supplementing our previous work with conventionally-prepared material. We studied virus particles just inside the nuclear envelope, between the leaflets of the nuclear membrane, and just outside the membrane at various stages of envelopment, and compared these particles to mature extracellular particles. Surface-rendered models of representative examples of virus particle egress in two stages (just inside the nuclear envelope, and just before egress from the nucleus) were made. Animation movies for presentation were also made.We saw a specialization of the inner leaflet of the nuclear envelope adjacent to, and conforming to, the icosohedral shape of an internal virus particle. We also saw evidence that the nuclear envelope is the origin of viral 'transport vesicles'. In several tomograms, we caught virus particles just inside the nuclear envelope, between the leaflets of the nuclear membrane, and just outside the membrane at various stages of envelopment, and compared these particles to mature extracellular particles.
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