Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells.

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.     

Replication of herpes simplex virus and cytomegalovirus in human leukocytes.

Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells II. An Ultrastructural Study of Viral Penetration

An electron microscope study was carried out on the early minutes of herpes simplex virus (HSV) and cytomegalovirus (CMV) penetration into WI-38 cells. Both HSV and CMV entered cells either by fusion of the viral envelope with a limiting cell membrane, or via phagocytosis. Both fusion and phagocytosis occurred within 3 min after the initiation of penetration. After fusion, the naked capsids of CMV free in the cytoplasm became coated with a fine, fibrillar material. CMV capsids thus coated retained a well-defined and easily identifiable morphology until the eclipse of visible viral particles between 1 and 1.5 days postinfection. In contrast, naked HSV capsids free in the cytoplasm were never coated. Rather, within minutes after penetration, they assumed a rounded, less regular outline, and were no longer detectable by 90 to 120 min postinfection. The free naked capsids of both viruses appeared to migrate across the cytoplasm toward the nucleus and to become located near nuclear pores. Both HSV and CMV capsids reached the nucleus as early as 5 min after the initiation of penetration. No further interaction with the nucleus could be documented. Particles were also consistently identified in the Golgi region. Phagocytosed particles generally remained within phagosomes, where they appeared to be degraded. However, stages were identified in what is believed to be the escape of enveloped viruses from phagosomes into the cytoplasm via fusion of their envelope with the phagosomal membrane.     

Specific immunity after congenital or neonatal infection with cytomegalovirus or herpes simplex virus

Infants or children who had congenital or neonatal infection with cytomegalovirus (CMV) or herpes simplex virus (HSV) have fewer than 1:30,000 mononuclear cells in their blood lymphocytes preparations that proliferate in cultures stimulated with the corresponding viral antigens. CMV and HSV responder cell frequencies in children and adults whose immunity followed postnatal infection with these viruses are 1:10,000 to 1:20,000. The low precursor frequency after congenital or neonatal infection is not associated with defective antigen processing by monocytes or nonspecific immunosuppression. Phenotypic changes in T cell subsets and the presence of antibody in the subjects suggests that the virus(es) do indeed elicit an immune response, but that this response is quantitatively deficient.     

Intracellular Traffic of Herpes Simplex Virus Glycoprotein gE: Characterization of the Sorting Signals Required for Its trans-Golgi Network Localization

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are two pathogenic human alphaherpesviruses whose intracellular assembly is thought to follow different pathways. VZV presumably acquires its envelope in the trans-Golgi network (TGN), and it has recently been shown that its major envelope glycoprotein, VZV-gE, accumulates in this compartment when expressed alone. In contrast, the envelopment of HSV has been proposed to occur at the inner nuclear membrane, although to which compartment the gE homolog (HSV-gE) is transported is unknown. For this reason, we have studied the intracellular traffic of HSV-gE and have found that this glycoprotein accumulates at steady state in the TGN, both when expressed from cloned cDNA and in HSV-infected cells. In addition, HSV-gE cycles between the TGN and the cell surface and requires a conserved tyrosine-containing motif within its cytoplasmic tail for proper trafficking. These results show that VZV-gE and HSV-gE have similar intracellular trafficking pathways, probably reflecting the presence of similar sorting signals in the cytoplasmic domains of both molecules, and suggest that the respective viruses, VZV and HSV, could use the same subcellular organelle, the TGN, for their envelopment.     

Diagnostic virology in a community hospital

Seven and one-half years of experience in a small diagnostic virology laboratory of a large inner-city hospital are reported. Seven hundred fifty-one viruses were isolated from over 8,000 specimens, using two types of tissue culture cells, human and monkey kidney. The most common isolates were Herpes simplex viruses (HSV) and Enteroviruses. Similar results have been reported by larger laboratories. Sensitivity for HSV in monkey kidney cells was only 75 percent that in human cells. An enzyme-linked immunosorbent assay (ELISA) for cytomegalovirus (CMV) was found to be a suitable substitute for the traditional complement fixation test (CF). IgM antibodies were not found in all HSV infections, but these antibodies did appear before CF antibodies in some cases. Monoclonal antibodies to HSV were effective in typing isolates, but for detection of viral antigen in brain smears of HSV encephalitis patients, polyclonal antibody gave better results.     

Replication of herpes simplex virus in two cell systems derived from rhesus monkeys

The replication of herpes simplex virus (HSV) in two cell systems derived from rhesus monkeys (LLC-MK2 and DBS-FRhL-2) was studied. In LLC-MK2, the growth of HSV-1 was abortive or extremely limited regardless of the multiplicity of infection, while that of HSV-2 was productive only on infection at high multiplicities. DBS-FRhL-2 cells supported growth of both types of HSV, although growth was highly dependent on the age of monolayers and the infectious dose of virus inocula. Plaques were produced in DBS-FRhL-2 cell monolayers inoculated with HSV-2 but not with HSV-1, although the efficiency of their formation in the former system was much less than in a system of FL and HSV-2. On the other hand, plaques were not produced in LLC-MK2 cell monolayers by either type of HSV. The growth of adapted variants of HSV-1 was also studied. In contrast to the parental strain, these variants replicated well in LLC-MK2 even at a low multiplicity of infection and produced clear plaques in the monolayers. Furthermore, persistent infections of HSV-2 were established in DBS-FRhL-2 cell monolayers under routine culture conditions.     

Cytoplasmic organization of POXvirus DNA replication

Poxviruses, a family of large DNA viruses, are unique among DNA viruses, because they carry out DNA replication in the cytoplasm rather than the nucleus. This process does not occur randomly, but instead, these viruses create cytoplasmic 'mini-nuclei', distinct sites that are surrounded by membranes derived from the rough endoplasmic reticulum (ER) that support viral replication. This review summarizes how distinct steps preceding cytoplasmic DNA replication, as well as replication itself, operate in the host cell. The collective data point to an important role for both the rough ER and the microtubules and indicate that these cellular structures help to co-ordinate the virus life cycle to ensure that individual steps occur at the right time and place. In a broader sense, they emphasize how viruses have evolved sophisticated ways to use host cells to optimize their life cycles to ensure efficient production of infectious progeny.     

S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function.

We have identified mutant strains of S. cerevisiae that fail to properly arrest their cell cycles at mitosis in response to the loss of microtubule function. New bud emergence and DNA replication (but not cytokinesis) occur with high efficiency in the mutants under conditions that inhibit these events in wild-type cells. The inability to halt cell cycle progression is specific for impaired microtubule function; the mutants respond normally to other cell cycle-blocking treatments. Under microtubule-disrupting conditions, the mutants neither achieve nor maintain the high level of histone H1 kinase activity characteristic of wild-type cells. Our studies have defined three genes required for normal cell cycle arrest. These findings are consistent with the existence of a surveillance system that halts the cell cycle in response to microtubule perturbation.     

Control models of cell cycle transit, exit, and arrest

Several distinct cycles mediate the events which occur between one cell division and the next. In micro-organisms there are generally two cycles. One governs biomass growth, the other DNA synthesis and cell division. In higher eukaryotes there can be as many as four distinct cycles, with growth, DNA synthesis, cell division, and nuclear division each possessing its own functional sequence of events. These cycles are controlled and coordinated by several different regulatory mechanisms. Restriction points are specific steps in the cycle whose completion is governed by external regulatory agents. One set of restriction points requires nutrients and growth hormones for step completion. Another set serves as receptors for differentiating factors which cause cycle arrest and initiate cellular differentiation. There is currently a debate as to whether restriction point inhibition involves permanent arrest or temporary arrest with a stochastic arrested-state residence time controlled by a transition probability mechanism. Tissue sizing is a process of negative feedback inhibition mediated by intercellular communication via cell surface contact and the extracellular matrix. Sizers commonly operate throughout broad portions of the cycle and appear to cause a slowing of cycle transit velocity rather than arrest. Sizers are probably the major regulatory mechanism for cell growth under conditions of nutrient and growth factor excess. They also generate compensatory proliferation following wounding or cell death. A growing body of evidence suggests that both the transit velocity, with which cells move through their several cycles, and the coordination of the cycles are controlled by intracellular regulatory mechanisms which behave as biological oscillators. These oscillators trigger complex sequences of events such as DNA synthesis and cell division.     

Hydroxyapatite for use as an animal cell culture substratum obtained by an alternate soaking process

Hydroxyapatite (HAp), the major inorganic component of hard tissues in vivo, was formed in/on a PVA gel. The HAp formation ratio depended on the reaction cycle number, but was independent of the alternate soaking period per cycle. The Ca/P molar ratio of HAp formed at 10 reaction cycles was very close to the theoretical value of HAp, 1.67. CHO-K1 cell adhesion, proliferation and maximum cell density on HAp plates were better on plates formed at two or five reaction cycles using 200 mM CaCl₂ and 120 mM Na₂HPO₄ solutions than on plates formed under other conditions. Furthermore, the adhesion ratio of CHO-K1 cells on HAp plates formed at 10 reaction cycles was about 60% of those at two or five reaction cycles.     

Herpes simplex virus type 1 infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b

We recently demonstrated that herpes simplex virus type 1 (HSV 1) induces a receptor on human umbilical vein endothelial cells for complement component C3b (C3bR). We assigned this receptor function to HSV 1 viral glycoprotein C (gC) based on several observations: tunicamycin, which prevents glycosylation and expression of N-linked glycoproteins on the surface of infected cells, markedly reduced expression of the C3bR; monoclonal antibodies to HSV 1 gC blocked detection of the C3bR, whereas monoclonal antibodies to other HSV 1 glycoproteins (gB, gD, gE) had no effect; and the MP mutant of HSV 1, which fails to express gC, did not induce C3bR. We now report that HSV 1 induces C3bR on a wide variety of cell types including bovine thoracic aorta and pulmonary artery endothelial cells, human embryonic lung and embryonic foreskin fibroblasts, and human embryonic kidney cells. To date, all cells studied that are permissive to HSV 1 express C3bR, although the pattern of rosetting of C3b-coated erythrocytes varies among the cell strains examined. We also demonstrate that C3bR expression is not a general response of human umbilical vein endothelial cells to injury, because three other viruses (adenovirus 7, measles, and mumps) do not induce C3bR after infection of these cells. Previously we had shown that among herpes simplex viruses, a variety of HSV 1 strains induce C3bR, whereas HSV 2 strains do not. We now demonstrate that other herpes family viruses (CMV and VZV) do not express C3bR. Therefore, C3bR expression appears to be unique for HSV 1 and occurs on a wide variety of cells permissive to this virus.     

DNA-tumor virus entry—From plasma membrane to the nucleus

DNA-tumor viruses comprise enveloped and non-enveloped agents that cause malignancies in a large variety of cell types and tissues by interfering with cell cycle control and immortalization. Those DNA-tumor viruses that replicate in the nucleus use cellular mechanisms to transport their genome and newly synthesized viral proteins into the nucleus. This requires cytoplasmic transport and nuclear import of their genome. Agents that employ this strategy include adenoviruses, hepadnaviruses, herpesviruses, and likely also papillomaviruses, and polyomaviruses, but not poxviruses which replicate in the cytoplasm. Here, we discuss how DNA-tumor viruses enter cells, take advantage of cytoplasmic transport, and import their DNA genome through the nuclear pore complex into the nucleus. Remarkably, nuclear import of incoming genomes does not necessarily follow the same pathways used by the structural proteins of the viruses during the replication and assembly phases of the viral life cycle. Understanding the mechanisms of DNA nuclear import can identify new pathways of cell regulation and anti-viral therapies.     

Characterization of the human newborn response to herpesvirus antigen

An investigation was made into the human newborn cellular response to herpes simplex virus type 1 (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV) to understand more about the nature of the neonate's susceptibility to overwhelming infection by these viruses. Newborn mononuclear cells sustained the proliferation in culture of maternal (i.e., haplotype-matched) T cell blasts with specificity for HSV, CMV, or VZV (p less than 0.05). This is evidence for intact antigen-processing capability by newborn monocytes. The response of the maternal T cell blasts appeared to be HLA-haplotype-restricted as suggested by experiments in which maternal T cell blasts were limited in number. Our culture conditions elicited responses predominantly from the T4+ lymphocyte subset. A low frequency of herpesvirus-specific T4+ lymphocytes in newborn blood might contribute to deficient viral immunity, so we evaluated the virus-specific T cell responding frequency in human newborns in limiting dilution cultures. We were unable to find a herpesvirus-specific responder cell frequency greater than 1:1,400,000 in nonimmune newborns. Three of seven adults who had no serum antibody to CMV had a CMV responder cell frequency (RCF) of 1:100,000 to 1:200,000. The RCF to HSV in immune children, ages 18 mo to 12 yr, and adults, ages 13 to 80 yr, ranged from 1:14,000 to 1:18,000. We conclude that newborn monocyte processing of herpesvirus antigen is intact, that T cell RCF is low in neonates, and that immunity to HSV after infection outside the newborn period results in comparable RCF between adults and children.     

Cytoplasmic phases in the first cell cycle of the activated frog egg

The first cell cycle of the activated frog egg is longer than subsequent cycles and several developmentally important events such as the determination of bilateral symmetry occur at this time. When eggs of Rana pipiens or Xenopus laevis are dissected at times after activation, differences in the consistency of the animal half cytoplasm can be detected visually, and the first cell cycle has been divided into four cytoplasmic phases on this basis. Phase 1 includes the events of activation and lasts about one-third of the first cycle. In phase 2, the cytoplasm becomes fluid except for the rigid, growing sperm aster, and most of the migration of the pronuclei occurs in phase 2. In phase 3, the cytoplasm becomes firm whether or not a sperm aster had been present, and the grey crescent forms, indicating the plane of bilateral symmetry. The firmness of the cytoplasm is colchicine sensitive but cytochalasin B insensitive as is grey crescent formation. In phase 4, the cortex detaches from the firm cytoplasm, and the firmness is now cytochalasin B sensitive and colchicine insensitive. The changes in cytoplasmic consistency during the first cell cycle probably reflect changes in the cytoskeleton, and the cytoplasmic consistency is functionally correlated with developmental events in the first cell cycle.     

Effect of arginine deficiency on the reproduction of human cytomegalovirus.

In arginine-deprived human embryonic fibroblasts the reproduction cycle of human cytomegalovirus (CMV) is incomplete. Infectious virus cannot be demonstrated in cell disintergrates, and from among the CMV antigens only the diffuse cytoplasmic antigen is detectable by immunofluorescence. The antigens localized in the cell membrane and those appearing during the complete cycle as large granules or inclusion-like bodies in the nucleus do not appear in the absence of arginine. The CMV genome persists in the arginine-deprived culture; after re-feeding with arginine-containing medium, maturation of virions soon ensues. Maturation could be prevented by inhibitors of protein synthesis, but not by DNA inhibitors, added simultaneously with completion of the medium.     

Production of good manufacturing practice-grade cytotoxic T lymphocytes specific for Epstein-Barr virus, cytomegalovirus and adenovirus to prevent or treat viral infections post-allogeneic hematopoietic stem cell transplant

Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.