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PubMed2003

Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids.

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.

Capsid↗
PubMed2000

Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major.

Protozoan parasites of the genus Leishmania undergo a complex life cycle involving transmission by biting sand flies and replication within mammalian macrophage phagolysosomes. A major component of the Leishmania surface coat is the glycosylphosphatidylinositol (GPI)-anchored polysaccharide called lipophosphoglycan (LPG). LPG has been proposed to play many roles in the infectious cycle, including protection against complement and oxidants, serving as the major ligand for macrophage adhesion, and as a key factor mitigating host responses by deactivation of macrophage signaling pathways. However, all structural domains of LPG are shared by other major surface or secretory products, providing a biochemical redundancy that compromises the ability of in vitro tests to establish whether LPG itself is a virulence factor. To study truly lpg(-) parasites, we generated Leishmania major lacking the gene LPG1 [encoding a putative galactofuranosyl (Gal(f)) transferase] by targeted gene disruption. The lpg1(-) parasites lacked LPG but contained normal levels of related glycoconjugates and GPI-anchored proteins. Infections of susceptible mice and macrophages in vitro showed that these lpg(-) Leishmania were highly attenuated. Significantly and in contrast to previous LPG mutants, reintroduction of LPG1 into the lpg(-) parasites restored virulence. Thus, genetic approaches allow dissection of the roles of this complex family of interrelated parasite virulence factors, and definitively establish the role of LPG itself as a parasite virulence factor. Because the lpg1(-) mutant continue to synthesize bulk GPI-anchored Gal(f)-containing glycolipids other than LPG, a second pathway distinct from the Golgi-associated LPG synthetic compartment must exist.

Animals↗
PubMed1988

Short chain fatty acids modulate nuclear receptor and extranuclear L-triiodothyronine levels in glial C6 cells by different mechanisms.

After incubation with [125]T3, the amount of radioactivity associated with the nuclear receptor and extranuclear compartment increases in glial C6 cells previously exposed to millimolar concentrations of butyrate. These actions of the fatty acid are accompanied by an inhibition of cell division, as shown by the decrease in [3H]thymidine incorporation or total DNA content per culture. Isobutyrate produced a similar effect at concentrations higher than 2 mM, whereas between 0.5-2 mM it increased the receptor content without affecting either extra-nuclear hormone or DNA. For both fatty acids there was a close parallelism between the dose-response curve for the inhibition of turnover of [3H]acetate from the histones and the increase in receptor levels. In contrast, a different dose dependence was found for the increase in extranuclear T3 levels, which suggests that the latter is caused by a mechanism other than histone acetylation. The increase in extranuclear T3 levels correlated better with the decrease in DNA and the accumulation of the chromosomal protein H10, which is thought to be related to the inhibition of cell proliferation. This suggests a relationship between the increase in extranuclear T3 and the inhibition of DNA synthesis. However, other agents, such as glucocorticoids or cAMP, which also inhibit C6 cell proliferation, were ineffective in increasing not only the receptor but also the extranuclear hormone. These findings show that the effect of short chain fatty acids on the receptor is not a consequence of the inhibition of cell replication, and that this inhibition could be related to, but is not sufficient per se for causing the increase in extranuclear T3.

Acetates↗
PubMed1998

Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.

After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex.

Cell Line↗
PubMed2005

An envelope-determined, pH-independent endocytic route of viral entry determines the susceptibility of human immunodeficiency virus type 1 (HIV-1) and HIV-2 to Lv2 restriction.

We identified a postentry restriction, termed Lv2, which determines the cellular tropism of two related human immunodeficiency virus type 2 (HIV-2) isolates and is dependent on the sequence of the capsid (CA) and envelope (Env) proteins. To explain the reliance on both CA and Env, we proposed that restrictive Envs deliver susceptible capsids to a compartment where Lv2 is active whereas nonrestrictive Envs deliver capsids into a compartment where Lv2 is either absent or less active. To test this model, we used compounds that affect endocytic pathways (ammonium chloride, bafilomycin A1, hypertonic sucrose) or lipid rafts (methyl-beta-cyclodextrin) to treat restrictive cells and show that restricted virus can be rescued from Lv2 if a lipid-raft-dependent, pH-independent endocytic pathway is inhibited. Furthermore, viral entry into HeLa/CD4 cells containing a tailless CD4 receptor, located outside lipid rafts, was fully permissive. Finally, we show that a variety of primary HIV-1 and HIV-2 viruses are susceptible to Lv2. Thus, we show that the route of entry, determined by the viral envelope, can influence cellular tropism by avoiding intracellular blocks to infection.

Ammonium Chloride↗
PubMed1999

HIV infection of the central nervous system is characterized by rapid turnover of viral RNA in cerebrospinal fluid.

To assess the kinetics of viral replication and decay in cerebrospinal fluid (CSF), we studied the short-term effects of highly active antiretroviral therapy (HAART) on CSF HIV-1 RNA concentrations. In 15 HIV-positive patients, HIV RNA concentrations were measured in paired CSF and plasma/serum samples. Samples were obtained prior to and 5 to 24 days after initiation or change of HAART. The short-term effects of interruption of HAART were tested in 2 patients. Viral load was measured by the Roche Amplicor assay. During HAART, in 12 of 15 patients a significant reduction of CSF HIV RNA concentration was observed, ranging from 0.55 to 2.77 log10 (median, 1.37 log10). This was paralleled by a reduction of blood viremia ranging from 0.12 to 3.0 log10 (median, 1.65 log10). The median half-life, as calculated from the slopes of the two time-point measurements, for CSF and blood viral load was 2.66 and 2.36 days, respectively. In 2 patients, CSF viral load remained essentially unchanged despite substantial reduction of plasma viral load. In 1 patient, after interruption of HAART, a rapid increase of HIV RNA in the CSF and blood was seen. No correlation was found between the CSF:blood albumin ratio as a measure of the functional integrity of the blood-CSF barrier and the ratio of CSF:blood RNA concentration, which suggests that no major passive influx of HIV RNA moves from the blood into the CSF compartment. However, a correlation existed between the CSF cell count and the CSF viral load (r = 0.74; p < .003). We conclude that, in most HIV-infected individuals, the decay of viral load in the CSF is similarly rapid as that seen in plasma. The rapid kinetics of virus found in the CSF suggest that it may be produced by rapidly proliferating cells, such as lymphocytes.

Adult↗
PubMed1988

[Use of cultured, virus-infected cells to study the biogenesis of polarity of epithelial cells].

The major characteristic of the eucaryote cell is the presence of specialized organelles in which macromolecular components responsible for various subcellular functions are segregated. The membranes of these organelles serve not only as divisions between the various cytoplasmic compartments, but also provide scaffolding within which the macromolecular complexes of the organelle assemble and become functionally integrated. It is obvious that because of the degree of complexity resulting from the existence of numerous compartments and membrane systems, the development of a genetic programme in a eucaryote cell requires not only the transcription of specific genes and translation in the cytoplasma of the resultant messenger RNA, but also the activity of mechanisms which ensure that each polypeptide reaches the site of its function, which may be in the cytosol, in a membrane, or in the luminal cavity of an organelle. In the special case of membrane proteins, such mechanisms must result not only in the specific distribution of polypeptides newly synthesized in the various types of cell membrane, but also the arrangement of them required in the lipid bi-layer necessary for their normal function.

Animals↗
PubMed1993

Adriamycin conjugates of human transferrin bind transferrin receptors and kill K562 and HL60 cells.

Adriamycin (ADR) was coupled to human transferrin (TRF) by using a glutaraldehyde crosslinking method. The TRF-ADR conjugates were separated by column chromatography and the molar ratio of ADR to TRF (i.e., conjugation number) for the studied conjugates was found to be 1.2. Analysis in sodium dodecyl sulfate-polyacrylamide gels demonstrated that TRF-ADR conjugates with this molar ratio had the same mobility as native TRF and contained few aggregates. The ADR remained conjugated to TRF under conditions of decreased pH known to occur in many intracellular compartments, and analysis by spectrofluorometry revealed that the conjugated ADR retained its ability to intercalate DNA. The TRF-ADR conjugates were shown by flow cytometry to preferentially bind tumor cells and cell-bound conjugates were found to be laterally mobile within plasma membranes. The binding of TRF-ADR conjugates was determined to be saturable, and competition experiments done with both radioiodinated and fluorescein-labeled TRF-ADR conjugates demonstrated dose-dependent inhibition of conjugate binding by unlabeled TRF, indicating that TRF-ADR conjugates were bound by TRF receptors. Cytotoxicity studies performed with tritiated thymidine incorporation and tetrazolium reduction assays revealed that TRF-ADR conjugates inhibited the proliferation of both K562 and HL60 cells in culture more effectively than free ADR. Such conjugates could provide a delivery system for ADR that would target the drug and possibly diminish its dose-associated complications.

Cell Survival↗
PubMed2000

EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency.

Infection of humans with Epstein-Barr virus (EBV) may cause infectious mononucleosis (IM). Analysis of single EBV-infected cells from tonsils of IM patients for rearranged immunoglobulin genes revealed two strategies of EBV for rapid and massive spread in the B cell compartment: the direct infection of many naive as well as memory and/or germinal center B cells and the expansion of the latter cells to large clones. In IM, the generation of virus-harboring memory B cells from naive B cells passing through a germinal center reaction likely plays no role. Members of clones can show distinct morphologies and likely also EBV gene expression patterns, and this ability implies a mechanism by which EBV-harboring cells can evade immune surveillance and establish a pool of persisting EBV-infected B cells.

Adolescent↗
PubMed1991

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.

Animals↗
PubMed1982

Acyclovir clinical pharmacology. An overview.

The current status of pharmacokinetic and toxicologic information on acyclovir is reviewed from a clinical pharmacologist's perspective. Acyclovir pharmacokinetics is accurately described by a two-compartment open model. The volume of distribution at steady state is about two-thirds of the body weight. The half-life of its beta phase of elimination is about three hours with normal renal function and increases to about 18 hours with anuria. Hemodialysis removes about 60 percent of the acyclovir in the body. The pharmacokinetics is independent of dose at least up to 15 mg/kg. Acyclovir is minimally (15 percent) protein-bound an only a small percentage (15 percent) of an intravenous dose is metabolized in persons with normal renal function. Acyclovir is eliminated primarily by glomerular filtration with a small addition from tubular secretion. The toxicity of acyclovir seems to be acceptably low. Local irritation with extravasation exists. Transient glomerular dysfunction is occasionally seen after bolus administration. Other side effects remain to be clearly established.

Acyclovir↗
PubMed2005

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Animals↗
PubMed1997

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.

Adenoviridae↗
PubMed2006

Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion.

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.

Bacterial Proteins↗
PubMed1994

Physical contact with lymphocytes is required for reactivation of dormant HIV-1 in colonic epithelial cells: involvement of the HIV-1 LTR.

HIV-1 transmission from mucosal epithelial cells to lymphocytes is a potential mechanism of HIV-1 contamination during sexual intercourse. The human colon epithelial cell line HT-29, that is infectable by various HIV-1 strains, is a useful model for studying the molecular mechanisms involved in this process. In the present study, we show that HT-29 cells, when exposed to either HIV-1(LAI) or HIV-1(NDK) at a low multiplicity of infection, became infected but did not produce infectious virions. Using two-compartment cell culture chambers separated by a porous membrane, we showed that PBL were able to rescue infectious HIV-1 from latently infected HT-29 cells following a physical interaction between the two cell populations. In contrast, HT-29 cells, infected with the same viruses at a high multiplicity of infection, were able to produce mature viral particles that were infectious to PBL in absence of cellular contacts. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that cell-to-cell contact induced an activation of the HIV-1 promoter. These observations provide a putative molecular mechanism for transmission of HIV-1 from mucosal epithelial cells to lymphocytes.

Cell Communication↗
PubMed2003

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions.

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.

Base Sequence↗
PubMed1997

[Clinical and flow cytometry outcome of improved photopheresis methods in dermatologic patients].

In photopheresis, 8-methoxypsoralen (8-MOP) is added to a mononuclear cell concentrate and then activated by UVA light, thus forming covalent bonds between DNA strands. Infusion of these modified cells that are not able to replicate any more seems to lead to the elimination of pathogenic T-cell clones and clinical improvement in patients suffering from cutaneous T-cell lymphoma, graft versus host reactions, and various autoimmune diseases. The original method described by Edelson et al. (1987) was improved by the following modifications proposed by Andreu et al. (1994): i) by using a cell separator (COBE Spectra) that produced purer concentrate with less red blood cells absorbing UVA light. ii) By applying 8-MOP directly into the collection bag, the drug side effects due to the oral application of the thousandfold dose and varying serum levels were avoided. iii) By irradiating the concentrate after collection, all cells received the same irradiation dosage. We treated 2 men with cutaneous T-cell lymphoma, 2 women with atopic eczema and 1 man with severe pustular psoriasis with overall 123 extracorporeal photochemotherapy (ECPC) sessions. In addition to routine laboratory analysis, detailed characterization of lymphocyte subpopulations was carried out by flow cytometry to differentiate T-helper and T-suppressor cells and their activation (HLA-DR, CD 25), B, NK cells, and monocyte subpopulations. The following mediators soluble were analyzed as well: Il-2-receptor and as indicators of acute phase reaction Il-8, neopterin and Il-1 beta. We observed a good clinical improvement independent of no significant trend in the immunological phenotype of circulating blood leukocytes. Our results suggest that ECPC effects are not mediated by a systemic immune response or alternatively are not measured in the blood compartment.

Dermatitis, Atopic↗
PubMed1988

Role of cytoplasmic vacuoles in varicella-zoster virus glycoprotein trafficking and virion envelopment.

Varicella-zoster virus (VZV) encodes several glycoproteins which are present on both mature viral envelopes and the surfaces of infected cell membranes. Mechanisms of VZV glycoprotein transport and virion envelopment were investigated by both continuous radiolabeling and pulse-chase analyses with tritiated fucose in VZV-infected cells. We studied in detail the large cytoplasmic vacuoles which were present in infected cells but absent from uninfected cells. The specific activity in each subcellular compartment was defined by quantitative electron microscope autoradiography, using a cross-fire probability matrix analysis to more accurately assess the individual compartment demarcated by the silver grains. By these techniques, we documented a progression of activity originating in the Golgi apparatus and traveling through the post-Golgi region into virus-induced cytoplasmic vacuoles and finally to areas of the cellular membrane associated with the egress of viral particles. Significant amounts of radiolabel were not observed in the nucleus, and only low levels of radiolabel were associated with the cellular membrane not involved with the egress of viral particles. In addition, immunolabeling of Lowicryl-embedded VZV-infected cells demonstrated the presence of VZV glycoproteins within cytoplasmic vacuole membranes as well as on virion envelopes. These observations suggested that cytoplasmic vacuoles harbored VZV-specified glycoproteins and were also the predominant site of VZV virion envelopment within the infected cell. Neither enveloped nor unenveloped viral particles were observed within the Golgi apparatus itself.

Autoradiography↗