Lysis of human arterial smooth muscle cells infected with herpesviridae by peripheral blood mononuclear cells: implications for atherosclerosis.

The cytotoxicity of peripheral blood mononuclear cells (PBMC) to human arterial smooth muscle cells (SMC) infected with cytomegalovirus (CMV) or herpes simplex virus-1 (HSV) was investigated. PBMC were isolated from heparinized blood of healthy donors by Ficoll-Hypaque centrifugation and were tested for cytotoxicity against human SMC or human fibroblast-like (MRC-5) cells infected with CMV or HSV, using the chromium-51 (51Cr) release cytotoxicity assay. Both SMC and MRC-5 cells infected with either CMV (SMC-CMV), (MRC-5-CMV), or HSV (SMC-HSV), (MRC-5-HSV) were lysed by PBMC above background lysis of uninfected SMC cells. Treatment of PBMC with NK-specific monoclonal CD16 antibody and rabbit complement reduced greatly the lysis of SMC, SMC-CMV, and K562 cells, suggesting that lysis of different types of target cell by PBMC was mediated mainly by natural killer (NK) cells. The pattern of natural cytotoxicity against SMC-CMV was different from that against SMC-HSV. Maximum lysis of SMC-CMV was observed at 24 hr postinfection compared to 8 hr postinfection for SMC-HSV. NK reactivity against SMC-CMV increased from 8 to 24 hr postinfection, followed by a gradual decline at 48 and 72 hr. Supernatants generated by culturing SMC-CMV or coculturing SMC-CMV with PBMC enhanced NK cell-mediated lysis of SMC or SMC-CMV. Natural cytotoxic reactivities of PBMC against SMC-CMV or SMC-HSV may occur in vivo. Such reactions could moderate the interaction of these viruses with vascular SMC and could influence the development and/or the progression of atherosclerotic lesions.

Characteristics of human arterial smooth muscle cell cultures infected with cytomegalovirus.

Distinct, sequential events occurring during the destruction and simultaneous regrowth of human arterial smooth muscle cell (SMC) cultures infected with cytomegalovirus (CMV, AD169 strain) were characterized. The events were influenced by the typical phenotypic diversity reflecting relative states of differentiation of the SMC cultures. Progenitors of regeneration were a surviving population of small, undifferentiated or relatively undifferentiated SMCs. As these cells reached confluence focally, the number of cells reactive with antismooth muscle serum, i.e. differentiating, increased, and in some postconfluent foci the organization of SMCs resembled the topography of uninfected cultures. Thus, infected SMC cultures had a limited capacity to repopulate, to organize typically, and to differentiate. However, continuing cytopathic effects gradually destroyed much of the regrowth, and a relatively large, nondividing SMC with prominent cytoplasmic filaments, similar to SMCs in terminal, uninfected cultures, predominated. Infected cultures consisting overwhelmingly of the large terminal phenotype were far less productive of infectious CMV than cultures populated by SMCs with continuing capacity to divide. Gradually, cultures consisting of the terminal phenotype deteriorated as a result of sporadic cytopathic effects of CMV and an effect resembling "senescent" degeneration in uninfected, nondividing cultures in late passage. The infected, terminal phenotype could be a latent or persistent source of CMV antigen or nucleic acid-positive cells detected by different investigators in normal and in atheromatous, human tissue, assuming that it exists and survives for an extended period in vivo after infection of vascular SMC.

Role of intranuclear sacs, intrasaccate tubes, and cytoplasmic dense bodies in the structural completion of cytomegalovirus.

The addition of tegument or matrix proteins and the outermost membrane to human cytomegalovirus (strain AD 169) replicating in human cells appeared to occur differently in the nucleus than in the cytoplasm. In the nucleus cytomegalovirus was completed structurally within intranuclear sacs, as envelopment at the end of tubular structures, containing an electron-dense material, included tegument and outer membrane simultaneously. In the cytoplasm structural completion occurred in separate steps. Tegument could be added by association with dense bodies; then, interaction with the membranes of vacuoles or vesicles provided the outermost covering. Tubes originating in intranuclear sacs have been described previously only in guinea pig cells infected with guinea pig cytomegalovirus.

Replication of cytomegalovirus in human arterial smooth muscle cells.

Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed.

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources.

The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg2+ to Mn2+ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV.

Concurrent replication of a papovavirus and a C-type virus in the CCL 33 porcine cell line.

A papovavirus, CCL 33 PV, isolated from a porcine cell line, CCL 33 (GT), was characterized. Based on a comparison of four isoenzyme systems, CCL 33 (GT) and CCL 33 (ATCC), obtained directly from the American Type Culture Collection, appeared to be the same. In addition to the previously characterized C-type virus of CCL 33 cultures, CCL 33 (GT) produced CCL 33 PV in high quantity, but CCL 33 (ATCC) produces a papovalike virus, presumably the same as CCL 33 PV, in barely detectable amounts. Serological results showed that CCL 33 PV is apparently identical to a papovavirus (SPV) isolated by Newman and Smith after inoculation of CCL 33 with concentrated porcine trypsin. These studies extend the characterization of this papovavirus, demonstrating that CCL 33 PV is weakly hemagglutinating after neuraminidase treatment, has a high affinity for a component of fetal bovine serum and is highly infectious in appropriate porcine cell systems rather than very defective as reported previously. Moreover, it was concluded from the data that CCL 33 PV is probably indigenous to the CCL 33 porcine cell line.

Reduction of herpes simplex virus-type 2 plaque formation by urea.

The effect of urea on plaque formation by herpes simplex virus type 2 (HSV-2) was examined in two systems at concentrations within or approximating the range found in human urine. Approximately 7--10 mg urea/ml, added 2, 4, or 8 hours after infection, reduced plaque formation by 50% in African green monkey kidney cells. The growth of this system was affected slightly by continuous treatment with urea at 7--10 mg/ml. Plaque formation was reduced in the monkey kidney system, albeit diminishingly, even after addition of urea 12 hours after infection. In the human lung fibroblast system, urea at 10 mg/ml reduced plaque numbers by 50% but depressed the growth of cells completely.