Viral alkaline nuclease in intranuclear dense bodies induced by herpes simplex infection

The distribution of the herpes simplex virus alkaline nuclease (HSV DNase) in rabbit fibroblast cells infected with HSV type 1 or 2 (HSV-1 or HSV-2) has been examined with the aid of immunocytochemical techniques using gold particles as markers. HSV DNase was found to be accumulated within infected nuclei as early as 2.5 hr post-infection. Labeled antibody to HSV DNase subsequently increased in all nuclei after 7 hr and 17 hr. At all times post-infection the virus induced nuclear dense bodies were always the most intensely labeled structures. The association of HSV DNase with nucleoprotein containing structures was readily dissociated by hypotonic shock and detergent treatment, but its association with the dense bodies was not disrupted. Nucleolar 100 kDa protein was found to be simultaneously present in the dense bodies. This suggests that HSV DNase might interfere with ribosomal RNA synthesis and play a role in the degradation of the host-cell metabolism.     

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.     

Localization of viral-specific 21 kDa protein in nucleoli of herpes simplex infected cells

Viral-encoded 21 kDa protein has been localized in herpes simplex virus type 1 (HSV-1) infected cells by immunocytochemical techniques using peroxidase and colloidal gold as markers. During early infection, 21 kDa protein was shown to be in cytoplasmic areas rich in ribosomes located near the nucleus and in the viral DNA-containing fibrillo-granular material of the virus-specific electron-translucent region in the nucleus. Late in infection, an additional marked accumulation occurred in both fibrillar and granular components of the nucleolus. Host chromatin and the nuclear dense bodies remained unlabeled. No immunolabeling was obtained in the absence of DNA replication. In contrast, inhibition of RNA synthesis did not modify the distribution of the protein. On the other hand, persistence of cytoplasmic and nuclear immunolabeling following the inhibition of protein synthesis performed late in infection indicated that the distribution of 21 kDa protein represented, at least in part, sites of accumulation and retention of preexisting molecules.     

Phosphorylation of the beta' Subunit of RNA Polymerase and Other Host Proteins upon phiCd1 Infection of Caulobacter crescentus

A protein kinase activity is induced early after infection of Caulobacter crescentus by the DNA phage Cd1. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. One of the phosphorylated DNA-binding proteins was identified as the β′ subunit of the host RNA polymerase.     

The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly in the cytoplasm (the perinuclear compartment). The P0 protein was found in the cytoplasm, as well as in the nucleus; however, the nucleoli were excluded. This protein was scattered around the nuclei, and the distribution might reflect association with the so-called nuclear bodies. This is the first example of r-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm.     

Association of two barley yellow mosaic virus (RNA 2) encoded proteins with cytoplasmic inclusion bodies revealed by immunogold localisation

Summary Antisera were raised against the RNA 2-encoded proteins of 28 kDa and 70 kDa of barley yellow mosaic virus (BaYMV) by using the corresponding cDNA sequences of a German isolate for protein overexpression inEscherichia coli BL 21 and subsequent purification. The proposed processing of a 98 kDa precursor polyprotein encoded by the long open reading frame of RNA 2 to two proteins of 28 kDa and 70 kDa could be confirmed by immunoprecipitation of the in vitro transcribed and translated cDNA-clone of RNA 2 and Western blot analysis of fragmentated protein extracts of BaYMV-infected winter barley plants. In situ localisation studies of infected leaf tissue using immunogold labeling techniques for electron microscopy revealed that both viral proteins of BaYMV (RNA 2) were associated with the crystal-like cytoplasmic inclusion bodies. No other parts of the cells and no other inclusions (pinwheelstructures or aggregated virus particles) showed any gold labeling when the 28 kDa and 70 kDa antisera were used. We suppose that both RNA 2-encoded proteins take part in the formation of the crystal-like cytoplasmic inclusion bodies which are the most dominant structures in the cytoplasm of BaYMV-infected tissue. Possible functions of the 28 kDa and 70 kDa protein of BaYMV (RNA 2) are discussed.Abbreviations PBS phosphate-buffered saline - CEA chicken egg albumin - BaYMV barley yellow mosaic virus - BaMMV barley mild mosaic virus     

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.     

Proteins and RNA in mouse L cell core nucleoli and nucleolar matrix

When intact nucleoli were prepared in the presence of enough leupeptin and phenylmethanesulfonyl fluoride to inhibit protease action, electrophoretic patterns of their constituent proteins were reproducible and very similar for L, HeLa, CHO, and rat hepatoma cells. "Core nucleoli", defined as that nucleolar fraction which remains after extensive DNase I action, had a protein composition similar to that of crude intact nucleoli, but were enriched for snRNA U3. Core nucleolar proteins included all of the histones, ribosomal proteins, and phosphorylated proteins with mobilities corresponding to 110 (protein C23) and 160 kilodaltons (kDa). The presence of protein C23 and of lamins A and C in nucleoli and core nucleoli was further verified by reaction with specific antibodies after one- or two-dimensional electrophoresis. A class of higher molecular weight proteins, ranging from 70 to greater than 200 kDa by mobility, was observed. It included at least 25 specific proteins, almost all of them highly acidic (pI less than 3.5). Treatment of core nucleoli with ethylenediaminetetraacetic acid/hypotonic buffer solubilized 30-35% of the small and large molecular weight proteins. In contrast, washing core nucleoli with 2 M NaCl selectively released U3 snRNA, 95% of the ribosomal RNA, and about half of the proteins, including C23 and most of the histones, ribosomal proteins, and other lower molecular weight proteins. The fraction remaining insoluble, "nucleolar matrix", was enriched for proteins of 34 and 57 kDa, lamins A and C, and most higher molecular weight proteins, as well as a portion of ribosomal spacer DNA.     

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.     

Possible mechanism of action of antiviral proteins from the leaves of Chenopodium album L

Antiviral proteins (AVPs) named CAP-I and CAP-II purified from the leaves of Chenopodium album cv Pusa Bathua-1 induced systemic resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in both hypersensitive as well as systemic hosts. An increased accumulation of two polypeptides (approximately 17 kDa and approximately 26 kDa) was observed in untreated upper leaves of Cyamopsis tetragonoloba plants whose basal leaves were treated with CAP-I/CAP-II. Both AVPs exhibited ribosomal RNA N-glycosidase activity on 28S rRNA of tobacco leaves and also caused in vitro degradation of TMV RNA. It is suggested that the CAP-I and -II are multi-functional and may be acting at multiple levels to ensure maximum possible inhibition of viral infection.     

"Reticular" and "Areticular" Nissl Bodies in Sympathetic Neurons of a Lizard

Sympathetic ganglia of the horned lizard, Phrynosoma cornutum, were fixed in OsO₄ and imbedded in methacrylate. Thin sections were cut for electron microscopy. Some adjacent thick sections were cut for light microscopy and were stained in acidified, dilute thionine both before and after digestion by RNase. In the light microscope two types of Nissl bodies are found, both removable by RNase: (1) a deep, diffuse, indistinctly bounded, metachromatic variety, and (2) a superficial, dense, sharply delimited, orthochromatic sort. Electron microscopically, the former ("reticular" Nissl bodies) corresponds to the granulated endoplasmic reticular structure of Nissl material previously described by others, whereas the latter ("areticular" Nissl bodies) comprises compact masses of particles of varying internal density and devoid of elements of endoplasmic reticulum. The constituent particles of the areticular Nissl material are 4 to 8 x the diameter of single ribonucleoprotein granules of the reticular Nissl substance and seem, near zones of junction with the reticular type, to arise by clustering of such granules with subsequent partial dispersion of the substance of the granules into an added, less dense material. It is suggested that the observed orthochromasia of the areticular Nissl substance is due to accumulation of a large amount of protein bound to RNA and, further, that these Nissl bodies may represent storage depots of RNA and protein.     

Sequestration of PML and Sp100 Proteins in an Intranuclear Viral Structure during Herpes Simplex Virus Type 1 Infection

We investigated the intranuclear distribution of PML and Sp100 in HeLa cells at the ultrastructural level and examined their relocalization in response to herpes simplex virus type 1 (HSV-1) infection. In the absence of infection, we observed that both are components, not only of nuclear bodies, but also of interchromatin granule-associated zones, which suggests a potential role for PML and Sp100 in splicing events. Prolonged HSV-1 infection induced dramatic changes in nuclear organization which consisted of the morphological disappearance of some nuclear structures (nuclear bodies, interchromatin granule-associated zones, coiled bodies) and of the development of a centrally located electron-translucent viral region which pushed the cellular clusters of interchromatin granules to the nuclear border. Concomitantly, dense bodies, concentric arrays of reduplicated inner nuclear membrane, and translucent patches containing a few viral capsids occurred at the nuclear border. PML and Sp100 were exclusively detected over the finely granular material of the vital translucent patches which also contains small amounts of p80-coilin and U1 and U2 snRNAs. An antiserum raised against capsid proteins intensely labeled the viral translucent patches at the level of their finely granular material and enclosed viral capsids. Our data, therefore, suggest that these viral structures, in addition to being the site of accumulation of vital capsid proteins and, possibly, a capsid-works, are also a site of sequestration of cell factors including PML and Sp100. Viral capsid proteins could interfere with and inactivate PML and Sp100 and be implicated in the shutoff of host cell metabolism induced by HSV-1 infection.     

The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells.

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Two RING Finger Proteins, the Oncoprotein PML and the Arenavirus Z Protein, Colocalize with the Nuclear Fraction of the Ribosomal P Proteins

The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.