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SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci-infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP-transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear "lamina" structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci., (© 2015 Mojica et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133- dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p-induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133- mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

A late gene encoding a 66-kDa structural protein of Autographa californica nuclear polyhedrosis virus was mapped and sequenced (26.9-29.7 MU). Transcription initiates from two conserved TAAG motifs (-15 and -37) with transcripts detected from 12 to 72 hr pi. The protein is detected in infected Spodoptera frugiperda (Sf9) cells from 24 to 72 hr pi. Western-blot and immunoelectron microscopic data identify this protein as specific for the polyhedra-derived virus (PDV) envelope. This protein has been named PDV-E66 to identify its viral origin, envelope location, and apparent molecular weight. In addition to being a structural protein of the PDV envelope, PDV-E66 is enriched in foci of microvesicles in the nuclei of infected Sf9 cells.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

Electron microscopic studies on the morphogenesis of SA8 in primary rabbit brain cell cultures revealed that in early stages of infection, envelopment of nucleocapsids commonly occurred at the inner nuclear membrane. From the perinuclear space, enveloped virus particles moved into the cisternae of the endoplasmic reticulum (ER) in which they were transported, through the cytoplasm, to the plasma membrane. Alternatively, de-envelopment at the outer nuclear membrane and egress of naked capsids into the cytoplasm were frequently observed. Non-enveloped cytoplasmic capsids were also a consistent feature of cells in late stages of infection, when nuclear membranes became ruptured. In these cases, the envelopment of naked capsids took place by budding either into the cisternae of ER or into cytoplasmic vesicles and vacuoles, in which transport to and exocytosis at the cell membrane occurred. Budding at the cell membrane was rarely found. Capsids of enveloped particles were asymmetrically surrounded by an electron-dense layer which may be identical to the tegument. Because only enveloped cytoplasmic and free virions were tegumented we suggested that the tegumentation must occur during the envelopment (budding) into cytoplasmic vesicles and at the plasma membrane.

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

The UL11 gene of herpes simplex virus 1 was reported to encode a myristylated protein (C. A. MacLean, B. Clark, and D. J. McGeoch, J. Gen. Virol. 70:3147-3157, 1989). To determine the function of the gene product, a recombinant virus (R7219) lacking 61% of the codons (176 bp of the 288-bp coding domain) was genetically engineered. The deletion mutant replicated in all cell lines tested, albeit to titers 30- to 250-fold lower than those obtained from cells infected with wild-type virus. Electron microscopic analyses indicated that both full and empty capsids accumulated in the nuclei, juxtaposed with the inner lamellae of the nuclear membranes, and that increased numbers of naked particles were present in the cytoplasm of cells infected with the mutant virus. There was a greater than 1,000-fold decrease in the amount of infectious extracellular virus released from Vero cells infected with the deletion mutant compared with that from cells infected with wild-type virus. Furthermore, the onset of release of infectious virus from cells infected with the UL11- mutant was significantly delayed: levels of extracellular UL11- virus increased 15-fold between 20 and 26 h after infection, while levels of wild-type extracellular virus increased 500-fold between 8 and 14 h after infection. A virus in which the UL11 gene was restored produced wild-type levels of total and extracellular virus and was indistinguishable from wild-type virus upon analysis by electron microscopy. Taken together, the data indicate that the absence of the UL11 gene causes a reduced capacity to envelope and transport virions into the extracellular space.

How the DNA tumor virus, simian virus 40, reaches the nucleus is unknown. In this report we have tested the affinity of simian virus 40 toward the nucleus by microinjecting virion particles into the cytoplasm under conditions in which cell-surface-mediated viral infection was blocked. Subcellular localization of viral structural proteins Vp1, Vp2, and Vp3, large tumor antigen, and virion particles was followed immunocytochemically and ultrastructurally. Both virion particles and viral structural proteins localized in the nucleus within 1-2 hr after cytoplasmic injection and subsequently expressed large tumor antigen, which was detected in the nucleus as early as 3 hr after cytoplasmic injection. Vp1 and large tumor antigen nuclear accumulation, as well as virion nuclear entry, were blocked by wheat germ agglutinin and an anti-nucleoporin monoclonal antibody, mAb 414. Virion particles were visualized in the vicinity of nuclear pores and in the cytoplasm with this agent. We conclude that virion particles are karyophilic and enter through nuclear pores. This study suggests that virion structural proteins facilitate virion import into the nucleus and viral gene expression.

The 72K immediate early (IE) 1 protein of human cytomegalovirus and the 68K protein, also encoded by the IE1 gene, were detected by immunoelectron microscopy using monoclonal antibodies specific for the 68K or 72K proteins. Early after infection, both proteins were localized to the nucleus, but with different localization patterns. Late after infection, both of the proteins decreased markedly in the nucleus, in which nucleocapsids appeared. Simultaneously, the 68K protein became diffusely distributed in the cytoplasm and the plasma membrane, whereas the 72K protein was distributed in the nuclear envelope and the plasma membrane. Both proteins were also observed in the coating membrane of the extracellular dense bodies.

The localization of adenovirus 2 (Ad2) and Minute Virus of Mice (MVM) DNAs was studied in situ in infected HeLa cell nuclei using fluorescent DNA probes and confocal microscopy. Ad2 DNA was found in multiple foci which were localized along the periphery of the infected cell nuclei. MVM DNA was found in HeLa cell nucleoli which are associated with the nuclear envelope, and when co-infected with Ad2 MVM DNA was compartmentalized to multiple foci which again were localized at the nuclear periphery. The data are discussed in terms of a model for the role of intranuclear compartmentalization in eukaryotic DNA structure and function.

An electron-microscopic investigation was made on hypodermal tissues of silkworms spontaneously infested by nuclear polyhedral disease, due to low temperature stress. It was established that the polyhedral virus is formed in the viroplasma produced after viral cell infection, is included in the intimate and developing membrane singly or in fascicles containing several (up to 9) and is finally incapsulated in polyhedral protein. Besides viral rods, various polymorphic forms were observed in the nucleus and around the polyhedras in the process of formation, which are considered as viral substructures (or nucleocapsidless viruses), membrane structures and surplus synthetized polyhedral protein. It can be presumed that virus replication in single cell nucleus is a continuous process which goes on until complete nuclear structure depletion.

Examination of brain tissue taken at autopsy from a 55-year old male showed the presence of herpes simplex viral antigen by immunofluorescence techniques. A suspension of this tissue was inoculated into cultures of human diploid fibroblasts. After 72 h, viral particles at different stages of development were seen inside the nuclei. Envelopment of the viral particles seemed to occur in association with the nuclear membranes, endoplasmic reticulum, cytoplasmic vacuoles and plasma membranes. Severely altered nuclear membranes, with as many as 4 to 8 layers of nuclear envelope, emphasized the common occurrence of membrane reduplication associated with herpes simplex infection. Unusual membrane-bound, dense, rounded inclusions, 100 to 500 nm in diameter, were present in the nucleus. These inclusions apparently were formed between the inner and outer lamellae of the nuclear membrane.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.

In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.

Cultures of cell line IZD-MB 0503 from Mamestra brassicae were inoculated with nonoccluded nuclear polyhedrosis virus of Autographa californica (AcNOV). At 1 h postinoculation (p.i.) nucleocapsids were found in the cytoplasm near nuclear pores and within the nucleoplasm. Formation of virogenic stroma was observed at 7 h p.i. The first short empty capsids were seen at 10 h postinoculation (p.i.), followed by partially and completely filled nucleocapsids (11-12 h p.i.) with most capsids filled at 12 h and later. This suggests that nucleocapsid components, such as capsid and DNA-core, assemble in successive stages rather than simultaneously. Viral progeny, being released from the cells by budding, were observed at 13 h p.i.

Immunoblotting with monoclonal as well as polyclonal lamin antibodies revealed that nuclear lamina proteins of human foreskin fibroblasts (HFF) were differentially affected after infection with human cytomegalovirus (HCMV). Lamin A immunoreactivity was progressively lost during the course of the infectious cycle whereas that of lamin C was comparatively stable. This process was not observed in herpes simplex virus-infected HFF. On the other hand, noninfected arrested HFF stimulated by serum to enter S-phase also exhibited loss of lamin A immunoreactivity. Selective in vivo proteolysis of lamin A is suggested as the possible underlying mechanism.

Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per micron2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.

Herpes simplex virus (HSV) type 1 was used to infect cultures of human embryonic dorsal root ganglion cells. Infected cultured were studied by electron microscopy. Viral nucleocapsids were observed to be internalized into neuronal cells bodies and neuritic extensions by fusion of the viral envelope and the plasma membrane. No signs of internalization by endocytosis were noted. Nucleocapsids were transported in neurites and were within 2 hrs postinfection found located near the microtubules and close to the nuclear pores in the perikaryon. A primary envelopment of nucleocapsids occurred at the inner lamina of the nuclear membrane and virions appeared between the two laminae. Presence of non-enveloped nucleocapsids outside the nuclear membrane and in close contact with the endoplasmic reticulum suggested that nucleocapsids could pass to the cytoplasmic side probably by de-envelopment at the outer nuclear membrane. A secondary envelopment occurred at the endoplasmic reticulum where the virions also became enclosed in transport vesicles. Enveloped virus appearing in the cytoplasm of neurons and neuritic extensions was always found only inside these transport vesicles. During their passage through the cytoplasm the virion-transport vesicle complexes were surrounded by smaller lysosome-like vesicles possibly derived from the Golgi apparatus. Fusion reactions between vesicles with virions and the smaller vesicles seemed to occur. We discuss if in this way the virion-transport vesicle complexes might be provided with glycosyl transferases and substrates necessary for maturation and completion of glycosylation of the viral envelope glycoproteins. The transport vesicles seemed essential for egress of virions from the infected cell by releasing virus when fusing with the plasma membrane.

Human diploid BAMB cells with epitheloid morphology, which had been derived from amniotic fluid cells, were capable of supporting the replication of human cytomegalovirus (CMV), without prior treatment of the cells with halogenated pyrimidines. The growth of this virus in BAMB cells and in human diploid fibroblastoid (LEP) cells was compared in parallel tests. Virus replication was slower and less efficient in the former than in the latter system. The most characteristic morphological feature of the CMV-infected BAMB cells was the formation of multinucleated giant cells which frequently contained more than a hundred nuclei; such cells were not seen in LEP cultures. The development of ultrastructural changes was slower in BAMB cells than in LEP cells. The additional most marked differences concerned the place of viral envelopment and the production of cytoplasmic dense bodies. While in LEP cells most nucleocapsids were enveloped from the inner leaflet of the nuclear membrane, in the other system a great majority of the particles acquired their envelopes by budding into vacuoles. Cytoplasmic dense bodies were rare in infected LEP cells but very frequent in BAMB cells. Budding of these structures into vacuoles was also observed.