Renee G. Traub

Interaction of the human polyomavirus, JCV, with human B-lymphocytes.

The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML). The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte. However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known. Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain. The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes. The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro. It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar.

Extension of JC virus host range to monkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatory region

A chimeric polyomavirus genome was constructed by inserting the 72- and the 21-bp repeats of simian virus 40 (SV40) into the JC virus (JCV) regulatory region on the late side of the JCV 98-bp repeats. Although the chimeric polyomavirus DNA was able to replicate very well in human fetal glial cells, deletions were found in sequences of the regulatory region. DNA sequence analysis of a selected clone indicated a 294-bp deletion from the original construction that retained the sequences for the JCV replication origin, 78 bp of one 98-bp repeat, 33 bp of one SV40 72-bp repeat, and one intact 72-bp repeat. Of significant interest was that this genome demonstrated an extended species and cell-type host range, producing infectious virus in human fetal brain and embryonic kidney as well as in rhesus monkey fetal and adult glial cells. However, the species host range was not extended beyond primate cells since the chimeric polyomavirus was unable to multiply in rodent glial cells. Analysis of the viral RNA transcripts from either kidney or glial cells indicated that the major start sites for early RNA mapped within the JCV sequences of the regulatory region and major start sites for late RNA mapped within the JCV and SV40 sequences. This extension of the JC virus host range was most likely attributable to changes in the regulatory region and not the viral T protein since a recombinant DNA clone which placed the coding sequences for the wild-type JCV T protein subject to regulation by the deleted chimeric regulatory region showed a similar extension of host range.

Expression of multiple classes of the nuclear factor-1 family in the developing human brain: differential expression of two classes of NF-1 genes.

Nuclear factor-1 (NF-1) is a multifunctional protein that participates in both transcription and replication. NF-1 proteins exist as a family of proteins that share some common structural and functional features but also demonstrate organ and cell type specific expression. Based upon these characteristics, the family of NF-1 proteins is divided into four classes, A, B, C and D. Several NF-1 binding sites have been identified in the regulatory sequences of the human polyomavirus, JCV, which multiplies most efficiently in glial cells derived from human fetal brain. Nuclear proteins from these cultures bind specifically to these NF-1 sites. It is not known, however, which member(s) of the NF-1 family is expressed in cells susceptible to JCV infection. We have examined glial cells as well as HeLa cells, which are not permissive to JCV, for NF-1 expression. By RT-PCR analysis, all four classes of NF-1 are expressed in human fetal glial cells and HeLa cells. However, by Northern analysis the expression of class D gene is much higher in the glial cells than HeLa cells. Expression of the class C gene, first identified in HeLa cells as NF-1/CTF1, is barely detectable in glial cells but highly expressed in HeLa cells. The screening of cDNA libraries from two early human brain tissues resulted in the identification of a number of clones which appear to be related and belong to a single class of the NF-1 family, class D. Nucleotide sequence of one clone, designated NF-1/AT1, confirms this. The NF-1/AT1 protein was overexpressed in E coli and found to bind specifically to an NF-1 probe by gel shift analysis. Southern analysis of human fetal glial cells indicates that the NF-1/AT1 gene, class D, is derived from a different gene than NF-1/CTF1. These results suggest the possibility that genes or viruses, like JCV, which use NF-1 for their expression in human brain derived cells may preferentially use the NF-1 class D protein.

Isolation of rubella virus from products of conception

Abstract The products of conception from 50 therapeutic abortions for maternal rubella in either the first or second trimester were studied using virus isolation procedures. Rubella virus was isolated from 32 (64 per cent) of the specimens. Within the limits of the study it could be demonstrated that the rubella virus was capable of infecting the products of conception, and that once infection was established the virus persisted within the products of conception as late as the seventy-seventh day following the onset of rash. For 8 infected fetuses it was possible to study individual organs. The placenta was involved in all 8 cases. In addition, isolations were achieved from brain, eye, inner ear, heart, thymus, lung, kidney, adrenal glands, gonads, spleen and the gastrointestinal tract in 2 cases, and from the heart in 1 case. This study suggests that the establishment of infection by the rubella virus within the placenta may be a very frequent phenomenon with maternal rubella in the first 4 months of gestation. The possible critical role of the placenta in determining whether infection of other fetal tissues occurs is discussed.

Evaluation of the role of cytokine activation in the multiplication of JC virus (JCV) in human fetal glial cells.

The human polyomavirus, JCV, is the etiologic agent of the fatal central nervous system demyelinating disease, progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy occurs most frequently in patients with underlying immunosuppressive disorders and is the direct result of virus multiplication in oligodendrocytes, the myelin producing cell in the central nervous system. In this report we test the ability of cellular activation signals to modulate expression of the JCV genome in either transfected or infected human fetal glial cells. In addition, we analyze the binding of nuclear proteins isolated from untreated and cytokine treated human fetal glial cells to transcription factor binding sites in the JCV regulatory region. In contrast to the effects of cellular activation on the expression of the HIV-1 promoter in these cells, none of the cellular activators tested increased expression of JCV. The cytokine, TNF-alpha, increased binding of NF kappa B (p50/p65) to a JC NF kappa B site but did not modulate the binding of nuclear proteins to the overlapping NF-1/AP1 region of the JCV enhancer. When taken together these results suggest that the response of JCV to cellular activation signals may be fundamentally different from the response of HIV-1 to these signals in human fetal glial cells and that the JC NF kappa B site may not be required for JCV gene expression or multiplication in vivo.

Some Characteristics of SSPE Measles Virus

Summary The in vitro comparison of two SSPE measles virus isolates and conventional measles virus demonstrated antigenic identity between these viruses. However, neutralization tests showed an avidity of the SSPE patients neutralizing antibody towards the wild measles virus rather than to the virus isolated from diseased brain. The cytopathology and plaque formation of these measles virus strains revealed greater resemblance between the SSPE measles and the vaccine strain of the virus than between SSPE measles and wild virus. Differences were found in the growth characteristics of the two SSPE virus isolates as measured by tissue culture assays.

Antibody Responses in Acute and Chronic Rubella

Summary Comparative antibody tests for rubella were conducted with the neutralization, indirect fluorescent, and complement fixation methods. All of these techniques demonstrated slightly different patterns of antibody response. Sero-conversion with infection was detected in almost all cases with the 3 methods. Antibodies detectable by fluorescent antibody and complement fixation tests decreased after 15 months following infection. By 10 to 20 years following rubella half of the CF tests were negative. Con-genitally infected children showed a decrease in antibody between the first and fifth month of age which was most marked in the CF test. Their full antibody response in these cases did not occur until after the sixth month.

JC virus T protein during productive infection in human fetal brain and kidney cells

We have examined the synthesis of the T protein of the human polyomavirus, JCV, during productive infection in primary cultures of human fetal glial and kidney cells. Immunoprecipitation of protein extracts from virus infected cells revealed that the JCV large T protein from both the prototype Mad and HEK adapted strains migrated as a 94-kDa protein in denaturing polyacrylamide gels. Resolution of the JCV T protein in brain cells could best be achieved following alkylation of the immunoprecipitated proteins prior to gel electrophoresis. The small t protein of either strain of JCV, however, could not be detected. In comparative experiments, the large T protein of the simian polyomavirus, SV40, was also identified as a 94-kDa protein in immortalized human fetal glial and kidney cultures. There were also protein complexes between p53 and SV40 T protein in the human glial and kidney cell lines. No evidence for a similar protein complex could be detected in JC virus infected human fetal brain or kidney cells.

The Etiology of Non-Specific Urethritis in Active Duty Marines

We studied patients with non-specific urethritis and control subjects at our dispensary. These patients and controls were matched for age, rank and sexual activity, and studied for the presence of bacteria, virus, Trichomonas and Mycoplasma. No significant bacteria were found in either group. No Trichomonas was identified, only 1 herpes II was recovered and no cytomegalovirus was found. Mycoplasma were cultured from patients and controls. The rate of colonization varied, depending upon several factors, but the significant factors seemed to be associated with the number of sexual partners. Those men with more than 1 sexual partner had a significantly increased colonization with Mycoplasma.

Cell Cultures from Human Fetal Brain Provide a Model for HIV-1 Persistence and Reactivation in the Central Nervous System

Cell cultures derived from human fetal tissues have been used for many years for the laboratory diagnosis of viral infections. In 1971, cultures prepared from human fetal brain were used in the isolation of a human polyomavirus, JCV, which was identified as the etiologic agent for the demyelinating disease, progressive multifocal leukoencephalopathy.1,2 Since JCV infected the oligodendrocyte in the brain, it was thought that JCV also infected the oligodendrocyte or its precursor cell in cultures from fetal brain.3,4 Laboratory investigations of the lineage of fetal brain-derived cells revealed that these precursor cells were difficult to identify and were in small numbers compared with other neural-derived cells, such as astrocytes and neurons.4–6 Since JCV does not infect neurons in the brain or in cultures, astrocytes were implicated as the predominant cell type in these cultures in which JCV multiplied.7 This observation was later confirmed both in situ in histopathological samples and in cell cultures with the establishment of astrocyte cell lines susceptible to JCV infection.8,9 Although cultures from fetal brain tissue demonstrate a diverse population of cell types, it has been possible to prepare cultures highly enriched for astrocytes. It has been these astrocyte-pure cultures from human fetal brain that have served as cell substrates for studies of neurotropism of viral pathogens. With the observation that HIV-1 was neuroinvasive (a characteristic of the lentivirus family), and was demonstrated in patients with dementia, laboratory and clinical investigations began to determine whether HIV-1 was also neurotropic, i.e., able to infect neural-derived cells. Astrocyte cell cultures from human fetal brain were used to initiate these studies.