Morphogenesis of human cytomegalovirus (HCMV) is

SummaryElectron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. Highpressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three-dimensional visualization after serial sectioning. For elucidation of dynamic processes threedimensional tomography is extremely helpful. We analysed interaction of HCMV particles with host cell membranes during final envelopment. Both wild-type virus and a viral mutant with impaired envelopment were analysed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) -a virus-induced cytosolic structure -by using threedimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.

Vaccines are one of the best health care advances ever developed, having led to the eradication of smallpox and near eradication of polio and diphtheria. While tremendously successful, traditional vaccines (i.e., whole-killed or live-attenuated) have been associated with some undesirable side effects, including everything from mild injection site inflammation to the autoimmune disease Guillain–Barré syndrome. This has led recent research to focus on developing subunit vaccines (i.e., protein, peptide, or DNA vaccines) since they are inherently safer because they deliver only the bioactive components necessary (i.e., antigens) to produce a protective immune response against the pathogen of interest. However, a major challenge in developing subunit vaccines is overcoming numerous biological barriers to effectively deliver the antigen to the secondary lymphoid organs where adaptive immune responses are orchestrated. Peptide amphiphile micelles are a class of biomaterials that have been shown to possess potent self-adjuvanting vaccine properties, but their optimization capacity and underlying immunostimulatory mechanism are not well understood. The present work investigated the influence of micelle size and charge on the materials’ bioactivity, including lymph node accumulation, cell uptake ability, and immunogenicity. The results generated provide considerable insight into how micelles exert their biological effects, yielding a micellar toolbox that can be exploited to either enhance or diminish host immune responses. This exciting development makes peptide amphiphile micelles an attractive candidate for both immune activation and suppression applications.

bWe report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb= region of the viral genome. A UL97-null (⌬97) mutant of strain TB40/E, which encodes a full-length ULb= region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a ⌬97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb= region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(␤-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb= regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ϳ100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb= regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb= region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb= region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

To sustain plant growth, development, and crop yield, sucrose must be transported from leaves to distant parts of the plant, such as seeds and roots. To identify genes that regulate sucrose accumulation and transport in maize (Zea mays), we isolated carbohydrate partitioning defective33 (cpd33), a recessive mutant that accumulated excess starch and soluble sugars in mature leaves. The cpd33 mutants also exhibited chlorosis in the leaf blades, greatly diminished plant growth, and reduced fertility. Cpd33 encodes a protein containing multiple C2 domains and transmembrane regions. Subcellular localization experiments showed the CPD33 protein localized to plasmodesmata (PD), the plasma membrane, and the endoplasmic reticulum. We also found that a loss-of-function mutant of the CPD33 homolog in Arabidopsis, QUIRKY, had a similar carbohydrate hyperaccumulation phenotype. Radioactively labeled sucrose transport assays showed that sucrose export was significantly lower in cpd33 mutant leaves relative to wild-type leaves. However, PD transport in the adaxial-abaxial direction was unaffected in cpd33 mutant leaves. Intriguingly, transmission electron microscopy revealed fewer PD at the companion cell-sieve element interface in mutant phloem tissue, providing a possible explanation for the reduced sucrose export in mutant leaves. Collectively, our results suggest that CPD33 functions to promote symplastic transport into sieve elements.

In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.

bThe product of the human cytomegalovirus (HCMV) UL71 gene is conserved throughout the herpesvirus family. During HCMV infection, protein pUL71 is required for efficient virion egress and is involved in the final steps of secondary envelopment leading to infectious viral particles. We found strong indications for oligomerization of pUL71 under native conditions when recombinant pUL71 was negatively stained and analyzed by electron microscopy. Oligomerization of pUL71 during infection was further verified by native and reducing polyacrylamide gel electrophoresis (PAGE). By in silico analyses of the pUL71 sequence, we noticed a basic leucine zipper (bZIP)-like domain, which might serve as an oligomerization domain. We demonstrated the requirement of the bZIP-like domain for pUL71 oligomerization by coimmunoprecipitation and bimolecular fluorescence complementation using a panel of pUL71 mutants. These studies revealed that the mutation of two leucine residues is sufficient to abrogate oligomerization but that intracellular localization of pUL71 was unaffected. To investigate the relevance of the bZIP domain in the viral context, recombinant viruses carrying mutations identical to those in the panel of pUL71 mutants were generated. bZIP-defective viral mutants showed impaired viral growth, a small-plaque phenotype, and an ultrastructural phenotype similar to that of the previously described UL71 stop mutant virus. The majority of virus particles within the viral assembly compartment exhibited various stages of incomplete envelopment, which is consistent with the growth defect for the bZIP mutants. From these data we conclude that the bZIP-like domain is required for oligomerization of pUL71, which seems to be essential for correct envelopment of HCMV.

; therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.T he production and release of infectious herpesviral particles is a multistep process that begins in the nucleus of the infected cell, where viral genomes are packaged into nucleocapsids. A recent study of pseudorabies virus (PrV) showed that the nuclear envelope breaks down to allow the capsid to exit the nucleus (23). However, many studies have shown that the widely accepted nuclear egress pathway requires capsid translocation through the nuclear membrane via primary envelopment and de-envelopment. This process is facilitated by a number of viral and host cell protein interactions. From the viral side, the two major proteins involved are the gene products of UL31 and UL34 of HSV-1 (pUL31 and pUL34, respectively) or their homologues in all other herpesviruses studied to date, which promote efficient primary capsid envelopment at the inner nuclear membrane (INM) (11,24,40,50). The pUL31 homologue of murine cytomegalovirus (MCMV), pM53, is distributed throughout the nucleosol in the absence of other viral proteins. pM53 is targeted to the INM after interacting with the membrane protein pM50 (the homologue of pUL34) via a mechanism that is conserved throughout herpesvirus morphogenesis (13,27,31,46,55,56,62). pM50 and pM53 form the nuclear envelopment complex (NEC) and recruit other cellular and viral proteins, such as protein kinase C and pUS3 (3,8,22,37,40,41,47,53), resulting in displacement of the rigid nuclear lamina and nucleocapsid budding (reviewed in references 19 and 36).The mechanism by which the pUL31 and pUL34 homologues mediate capsid transition through the nuclear envelope is still not fully understood, but disruption of either partner usually leads to retention of viral capsids within the nucleus (5,11,13,24,31,50,64). Insights into this mechanism were gained from studying the PrV proteins pUL31 and pUL34, which induce vesicle formation in the nuclei of transfected cells in the absence of other viral components (20), supporting their role in the capsid envelopment process at the INM. The reciprocal binding sites, which result in NEC targeting to the INM, are well characterized in pM53 and pM50 and are conserved throughout the pUL31 family (5,13,14,30,31,54,56). Furthermore, recent studies of HSV-1 suggest a second essential interaction between the N-term...