Focal nuclear envelope lesions and specific nuclear lamin A/C dephosphorylation during infection with human cytomegalovirus.

Morphological analysis of cytomegalovirus-infected human fibroblasts reveals characteristic alterations of the nuclear envelope during budding of the nucleocapsids from the nucleus. These changes include focal nuclear lamina thickening and formation of "blebs" at the nuclear membranes. Using a specific monoclonal antibody that recognizes a phosphorylated epitope in nuclear lamins A/C, we show here that the cytopathological alterations at the neighborhood of the nuclear lamina are paralleled by a process of systemic dephosphorylation of the nuclear lamins.

Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes.

We have investigated by electron microscopy the envelopment of progeny human cytomegalovirus particles and vaccinia virus particles. As host cells we used primary human foreskin fibroblasts, in which both viruses replicate, and also AtT20 cells in which vaccinia virus, but not human cytomegalovirus, replicates. As we show here, primary human foreskin fibroblasts contain tubular early endosomes like those in AtT20 cells and many other cells in culture. Our aim was to ascertain whether or not the cisternae which wrap the maturing progeny viral particles are related to tubular endosomes. When infected cells were incubated with the fluid phase endocytic tracer horseradish peroxidase (HRP) at appropriate times after infection (5-8 h post vaccinia infection and 72 h or 96 h post cytomegalovirus infection), we found that many of the partially or fully enveloped vaccinia and human cytomegalovirus particles in the cytoplasm had HRP reaction product in the lumen of their cisternal envelope. Examination of single and serial sections indicated that the cisternal envelopes of the progeny virions were derived from tubular endosomes. Pulse-chase experiments with HRP showed that the viral particles with envelopes derived from tubular endosomes were not directed to late endosomes and autophagic digestion but were released from the cells. Experiments with Brefeldin A established that the envelopment of viral particles by endosomal cisternae proceeded for at least 2 h in the absence of a Golgi apparatus. Our data indicate that vaccinia virus and human cytomegalovirus (and presumably other pox and herpes viruses) have evolved to utilize early endocytic compartments to achieve their egress from host cells. We also found that in cells infected by vaccinia virus the delivery of the endocytic tracer to the Golgi apparatus is greatly enhanced over that in controls or cells infected with human cytomegalovirus. The cytopathic effects of vaccinia virus therefore include perturbation of membrane traffic between endocytic and exocytic compartments.

Targeting of the gene product encoded by ORF UL56 of human cytomegalovirus into viral replication centers.

The highly conserved DNA-binding protein pUL56 of human cytomegalovirus (HCMV) was found to be predominantly localized throughout the nucleus as well as in viral replication centers of infected cells. The latter localization was abolished by phosphono acetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL56 co-localized in replication centers alongside pUL112-113 and pUL44 at late times of infection. By co-immunoprecipitations, a direct interaction with pUL44, a protein of the replication fork, was detected. These results showed for the first time that HCMV pUL56 is localized in viral replication centers, implicating that DNA replication is coupled with packaging.

Persistent long-term changes in lymphocyte subsets induced by polyclonal antibodies.

BACKGROUND: Clinicians are well aware of the short-term effects of immunosuppression by mono- or polyclonal antibodies. Little is known about long-term changes induced by these therapies. METHODS: Forty-three renal allograft recipients were selected according to their initial postoperative immunosuppression: (1) BI group=basic immunosuppression with steroids and cyclosporine, n=16; (2) ATG group=basic immunosuppression plus polyclonal antibody antithymocyte globulin (ATG), n=11; and (3) OKT3 group=basic immunosuppression plus monoclonal antibody OKT3, n=16 patients. At intervals of 6 months, the following parameters were measured prospectively: lymphocyte surface antigens (HLA-DR, CD3, CD4, CD8, CD16, CD19, CD56, and CD57); serum and urine neopterin; serum amyloid A; and indirect and direct tests for herpes viruses. RESULTS: The mean period of observation was 58.4 months. The most significant differences between the groups occurred for CD4+ and CD8+ T cells. The ratios of CD4+ to CD8+ cells (n=278 measurements) were significantly and persistently lower in the ATG group (P<0.001, Brown-Mood test). Five years after transplantation, the ATG group had a CD4+ to CD8+ cell ratio of x=0.6 versus x=1.7 in the OKT3 group and x=2.0 in the BI group. This inversion was due to a persistent depletion of the CD4+ cells and an increased regeneration of the CD8+ cells, in particular of the CD8+brightCD57+ subpopulation. Extent and duration of CD4+ depletion correlated with the cumulative ATG dose (r=0.7, P<0.05, Spearman rank correlation test). CONCLUSION: Therapy with polyclonal antibody ATG induces dose-dependent long-term changes in T-cell lymphocyte subsets, which persist over a period of years.

The UL112/113 gene products of human cytomegalovirus which colocalize with viral DNA in infected cell nuclei are related to efficient viral DNA replication.

The UL112/113 gene products of human cytomegalovirus (HCMV) were shown by transient complementation ori Lyt-dependent DNA replication assay to be early viral proteins required for efficient viral DNA synthesis. By immunofluorescence analysis followed by fluorescence in situ hybridization, we showed that UL112/113 gene products of HCMV are colocalized with viral DNA prior to and during viral DNA replication in infected cell nuclei. We have used an anti-sense RNA approach for functional analysis of the UL112/113 gene in HCMV. The astrocytoma cell line U373-MG was used for permanent expression of the anti-sense UL112/113 gene. Expression of the anti-sense RNA in this cell line significantly blocked expression of UL112/113 gene products and viral DNA replication, indicating that the UL112/113 gene products are related to efficient viral DNA replication.

Constitutive expression of human cytomegalovirus (HCMV) glycoprotein gpUL75 (gH) in astrocytoma cells: a study of the specific humoral immune response.

The humoral immune response to gpUL75 (gH) was determined in different groups of human cytomegalovirus (HCMV) infected subjects using a full-length glycoprotein constitutively expressed in an astrocytoma cell line. The recombinant molecule consisted of two distinct isoforms resembling the authentic protein of infected cells. Separated from the interactions of other viral gene products gH failed to form an oligomeric complex, thus exhibiting exclusively epitopes present on the monomer. Ninety five percent of serum samples from latently-infected healthy adults revealed the presence of gH-specific IgG. Moreover, examination of sequential sera from immunocompromised and immunocompetent individuals undergoing active HCMV infection demonstrated that antibodies to gH occurred in most cases simultaneously with those to the abundant surface antigen gpUL55 (gB) and at similar titres. Appearance of this response was correlated with a considerable increase of the virus-neutralizing activity and most likely associated with restriction of viral dissemination during subsequent viremic episodes. Together, these results suggest that glycoprotein H of HCMV is like gB, a highly immunogenic component of the infectious particle.

Assessment of cytomegalovirus infections using neopterin and a new immunoblot.

Human cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality in immunocompromised patients despite advances in diagnostic tests and antiviral therapies. The underlying study investigates the diagnostic value of the immune marker neopterin and a recently developed HCMV-specific western blot to detect HCMV infections and to differentiate them into either syndromes or diseases. The mean period of observation was 1428 days. Thirteen HCMV diseases and nine syndromes were diagnosed retrospectively. The first appearance of clinical signs or symptoms was always associated with a marked increase of serum and urine neopterin. The HCMV-specific IgM response followed in the mean 9 days later. Median values and the course of the neopterin levels were significantly higher during the HCMV diseases. In addition, the strength of the humoral immune response was related to the severity of the HCMV infection. Patients with HCMV diseases developed antibodies against a higher number of epitopes. The anti-HCMV IgM response persisted in more than 80% of the patients for longer than 3 years. In conclusion, combining the HCMV-specific western blot and neopterin permit detection of the immune response against HCMV, reflect the severity of the infection and might guide the anti-viral therapy.

Sodium butyrate selectively inhibits host cell glycoprotein synthesis in human fibroblasts infected with cytomegalovirus.

Host cell as well as viral DNA synthesis in human fibroblasts infected with human cytomegalovirus was found to be largely resistant even to high concentrations of sodium butyrate. Likewise, production of viral progeny was reduced by 1-2 orders of magnitude but not abolished. On the other hand, the drug allowed (modified) glycosylation only of viral polypeptides whereas that of host proteins was suppressed. Immunofluorescence studies on living cells suggested that butyrate may interfere with processing and intracellular transport of virus-specific surface membrane antigens.

Site directed mutagenesis of the carboxyl terminus of human cytomegalovirus glycoprotein B leads to attenuation of viral growth in cell culture.

A viable human cytomegalovirus (HCMV) mutant was generated harbouring a glycoprotein B (gB) in which the carboxyl-terminal amino acids DRLRHR (aa 885-900) were changed to AALREE. Characterization of the phenotype of the recombinant virus revealed significant reduction of infectious progeny release and only moderate reduction of viral DNA replication indicating its diminished specific infectivity. This observation was in line with immunogold labeling of extracellular virions demonstrating that the amount of gB protein was markedly reduced in the envelope of the mutant virus. Our results suggest that the conserved carboxyl-terminus of the gB molecule is critical for HCMV maturation.

Effect of herpes simplex virus type 1 infection on the cellular DNA polymerase activities of mouse cell cultures.

Thymidine kinase-deficient mouse cell cultures infected with herpes simplex virus type 1 exhibited a maximum of virus DNA synthesis around 8 h post-infection as determined by pulse labelling with 3H-thymidine. Cellular DNA synthesis was progressively inhibited, but still appreciable until 8 h post-infection and not completely abolished at any time during the infectious cycle. Phosphonoacetic acid was found to be a potent and selective inhibitor of virus DNA synthesis only when added to infected cultures before the onset of virus DNA synthesis. During the interval of increasing virus DNA synthesis the activity of cellular alpha polymerase decreased rapidly, whereas the beta polymerase activity increased significantly; a slight increase was observed for the gamma polymerase activity. When infected cells were kept in the presence of phosphonoacetic acid following virus adsorption the effect on cellular DNA polymerases was less pronounced.

Unimpaired histone synthesis in human fibroblasts infected by human cytomegalovirus.

Serum-starved human foreskin fibroblasts were infected by human cytomegalovirus (Towne strain) that is thought to induce DNA replication in host cells during lytic infection. At various times postinfection, the cultures were pulse labeled with either 3H-thymidine or 14C-thymidine and 3H-lysine to examine DNA synthesis and histone synthesis, respectively. Isopycnic centrifugation of labeled DNA in CsCl revealed that precursor incorporation into host-cell DNA was enhanced over the control around 24 h postinfection and decreased after onset of viral DNA synthesis which reached a peak around 72 h postinfection. For analysis of histones 3H-lysine-labeled proteins of lysates of unfractionated cells and of chromatin preparations were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and subsequent fluorography. Comparison of the fluorograms from the various pulses postinfection suggested that 3H-lysine incorporation into histones exhibited no major variations concurrent with the changes of host-cell DNA synthesis. In contrast, herpes simplex virus type 1 was found progressively to extinguish histone synthesis in the course of the cellular infection. Furthermore, histone synthesis in phosphonoacetic acid-treated cytomegalovirus-infected cultures was not enhanced over that in mock-infected controls. These observations do not support the view that human cytomegalovirus induces host-cell DNA replication under the conditions used.

Changes of mitochondrial DNA polymerase-gamma activity in synchronized mouse cell cultures.

Following synchronization by a double hydroxyurea block, mouse cell cultures exhibited a period of accelerated precursor incorporation into mitochondrial DNA during the late nuclear S phase. Peak activity of mitochondrial DNA polymerase-gamma occurred concurrent to the interval of accelerated organelle DNA synthesis. Mixing experiments suggested that the variations in mitochondrial DNA polymerase activity during the cell cycle were not due to free inhibitors in the enzyme preparations examined.

Glycosphingolipid synthesis in human fibroblasts infected by cytomegalovirus.

Infection of serum-deprived human foreskin fibroblasts (HFF-cells) by cytomegalovirus (HCMV) resulted in enhanced precursor incorporation into glycosphingolipids (GSL). Analysis of the component patterns revealed a unique biphasic effect on the rate labeling of neutral GSL. Early during infection or in the presence of phosphonoacetic acid, radiolabel was found predominantly in ceramide tri- and tetrahexoside, whereas late after infection label in ceramide monohexoside exceeded that in the other components. Determination of the chemical amounts of neutral GSL components from infected cultures supported the view of increased biosynthesis of ceramide tri- and tetrahexoside early, and of ceramide monohexoside late postinfection. Changes, comparable to those observed under the influence of "early" viral functions were observed also during S-phase of serum-stimulated HFF cells. With respect to acidic GSL a decrease of metabolic labeling occurred late during infection. Relatively little alteration was found in the component pattern.

Chloramphenicol-induced loss of mitochondrial DNA polymerase activity in HeLa cells.

HeLa cells exposed to chloramphenicol for approximately one cell generation were found to contain a mitochondria-associated DNA polymerase with a significantly lower specific activity than that of control cells. This observation was not due to the presence of inhibitors in mitochondrial DNA polymerase preparations of chloramphenicol-treated cell cultures. In addition, there was no accumulation of a typical mitochondrial DNA polymerase in the post-mitochondrial supernatant of drug-treated cells.