Analysis by in situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Low-icryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

Analysis by in Situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Lowicryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

Bismuth staining of a nucleolar protein

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.     

Detection by bismuth staining of highly phosphorylated nucleo-proteins in plants. Determination of its specificity by X-ray microanalysis,SDS-PAGE and immunological analysis

Summary— Staining with bismuth salts after glutaraldehyde fixation is a very useful technique for preferential detection of phosphorylated nucleoproteins in mammalians and insects. In the present work we report an adaptation of this method for plant nuclei: staining with bismuth salts either in tissue blocks before embedding, or on thin sections of acrylic resin. Both procedures are highly reproducible and give the same pattern of staining in the nuclei in situ or isolated at the electron microscope. The specificity of bismuth binding to the dense nucleolar fibrillar component and interchromatin granules is proven by X-ray microanalysis. The nuclear proteins which bind bismuth have been identified by bismuth and immunostains of blots from total nuclear proteins. This technique is a very useful and specific cytochemical tool for studying nuclear organization and functions in plants.     

Herpes simplex virus type 1-induced modifications in the distribution of nucleolar B-36 protein

Nucleolar B-36 protein was localized ultrastructurally by immunocytochemistry with monoclonal antibody P2G3 and colloidal gold label in rabbit fibroblast cells before and during infection with herpes simplex virus (HSV) type 1. In non-infected cells, labeling was sparse and restricted to the fibrillar component of the nucleoli. During the infectious cycle, B-36 protein appeared to be somewhat more abundant within the morphologically altered fibrillar component of the nucleoli. In addition, the protein was also detected in some but not all virus-induced intranuclear dense bodies. These observations suggest the presence of functionally distinct dense bodies. The association of B-36 protein with both structures was not disrupted by a hypotonic shock and detergent treatment, which suggest that these sites do not represent areas of passive intranuclear diffusion. Inhibition of protein synthesis late in infection, viral DNA replication or RNA synthesis did not alter the distribution of B-36 protein. We suggest that this protein may play a role in the increased compaction of the ribonucleoprotein fibrils induced by HSV infection, perhaps in association with some of the virus-encoded proteins which also have been detected in the nucleoli.     

Ultrastructural and X-ray analytical studies on intranuclear bismuth inclusions

Parenteral administration of bismuth subnitrate produced intranuclear inclusions in the rabbit kidney tubules. In glutaraldehyde fixed material these inclusions had a homogeneous appearance but in osmicated material some of these inclusions showed a granular and fibrillar substructure. In relatively thick unstained sections prepared from unosmicated tissues, it was found that the inclusions were electron-dense, but they vanished from view when the electron beam was focussed on them. This phenomenon is thought to be due to the presence of bismuth, which is a mental with a high electrical resistance and low thermal conductivity. With the aid of electron-probe analysis the presence of bismuth was demonstrated in these inclusions and it seems possible that sulphur may be present here.     

Interactions of minute virus of mice and adenovirus with host nucleoli.

Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation.     

Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein

SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.     

Deubiquitinating function of adenovirus proteinase

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.     

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.     

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis.

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosome-phase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late cap-phase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.     

Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde-fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non-infected cells. From 2.5-17 hr post-infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus-modified pre-existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus-induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent-induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.     

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.     

Bismuth staining for light and electron microscopy.

Bismuth salts on aldehyde fixed tissue give a highly selective pattern of staining suitable for light and electron microscopy. Structures stained include the nucleolus, ribosomes, inter- and perichromatin granules, the Golgi complex beads and the outer face of the tubule doublets of mouse sperm, certain neurosecretory vesicles believed to contain biogenic amines, some junctions (some central synapses, neuromuscular junctions, tight junctions), specialized membranes such as the post acrosomal dense lamina of mouse sperm and the inner alveolar membrane of Paramecium, and a variety of structures associated with the cytoplasmic face of membranes, such as plasma membrane plaques, cleavage furrows, the leading edge of the spreading acrosome and sperm annuli.Staining is not reduced by nucleases and spot tests show no reaction between nucleic acids and bismuth under conditions similar to those used to stain tissues. However, spot tests do show strong binding of bismuth by basic proteins and by some phosphorylated molecules.It is hypothesized that bismuth reacts with cell components in two ways, distinguishable by their glutaraldehyde sensitivity. For example, staining of the nucleolus and ribosomes is blocked by glutaraldehyde but the inter- and perichromatin granules and the GC beads are unaffected. Spot tests show that basic proteins (histones, protamines, polylysine and polyargenine) and other molecules with free amino groups (5HT, tryptamine, dopamine) bind bismuth strongly, a reaction that is blocked to varying degrees by glutaraldehyde. We presume that most bismuth staining of tissues is due to reaction with amine groups and is glutaraldehyde sensitive and some may be due to guanidine groups which are less sensitive to fixation by glutaraldehyde. Organic phosphates may be the cause of the glutaraldehyde insensitive staining since ATP and some other phosphates bind bismuth in a reaction that is not blocked by glutaraldehyde.     

Characterization of adenovirus-induced inverted terminal repeat-independent amplification of integrated adeno-associated virus rep-cap sequences

Stable packaging cell lines expressing the rep and cap genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa rep-cap cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the inverted terminal repeats (ITRs) deleted. We now report a more detailed analysis of this phenomenon and highlight the key cellular and viral factors involved. Determination of the rep-cap copy number of HeRC32 cells indicated that maximum rep-cap amplification occurred between 24 and 48 h following adenovirus infection. Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified rep-cap sequences were found in an extrachromosomal form. Amplification of the rep-cap sequence with the ITRs deleted was not dependent on adenovirus replication and still occurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indicating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was essential, since rep-cap amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant encoding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of rep-cap amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in rep-cap amplification. Indeed, the lack of detectable amplification in an adenovirus-infected stable rep-cap HeLa cell clone unable to produce Rep proteins further supported that, among the viral gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated rep-cap sequences in the absence of the AAV ITRs.