STRL33, A novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1.

The chemokine receptors CXCR4, CCR2B, CCR3, and CCR5 have recently been shown to serve along with CD4 as coreceptors for HIV-1. The tropisms of HIV-1 strains for subgroups of CD4(+) cells can be explained, at least partly, by the selective use of G protein-coupled receptors (GPCRs). We have identified a novel human gene, STRL33, located on chromosome 3 that encodes a GPCR with sequence similarity to chemokine receptors and to chemokine receptor-like orphan receptors. STRL33 is expressed in lymphoid tissues and activated T cells, and is induced in activated peripheral blood lymphocytes. When transfected into nonhuman NIH 3T3 cells expressing human CD4, the STRL33 cDNA rendered these cells competent to fuse with cells expressing HIV-1 envelope glycoproteins (Envs). Of greatest interest, STRL33, in contrast with CXCR4 or CCR5, was able to function as a cofactor for fusion mediated by Envs from both T cell line-tropic and macrophage-tropic HIV-1 strains. STRL33-transfected Jurkat cell lines also supported enhanced productive infection with HIV-1 compared with control Jurkat cells. Despite the sequence similarities between STRL33 and chemokine receptors, STRL33-transfected cell lines did not respond to any in a panel of chemokines. Based on the pattern of tissue expression of the STRL33 mRNA, and given the ability of STRL33 to function with Envs of differing tropisms, STRL33 may play a role in the establishment and/or progression of HIV-1 infection.

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter.

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Isolation of mutants of an animal virus in bacteria.

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.

SV40 large T antigen with c-Jun down-regulates myelin P0 gene expression: a mechanism for papovaviral T antigen-mediated demyelination.

Expression of myelin proteins has been shown to be altered in transgenic mice that express papovaviral large tumor (T) antigens. This paper analyzes the effect on P0 gene expression in secondary Schwann cells transfected with the SV40 T antigen gene and in Schwann cells immortalized by T antigen. In secondary Schwann cells, both T antigen and c-Jun are required for significant inhibition of the P0 promoter; expression of only one of the proteins is insufficient for repression of the P0 gene. T antigen, c-Jun (p39), and c-Jun-related protein (p47) form an immunoprecipitable complex in SV40 immortalized Schwann cell lines, and T antigen and c-Jun bind independently and as a complex to the P0 promoter. Our data suggest that the probable molecular mechanism underlying the hypomyelination observed in transgenic animals expressing T antigen may be due to the repression of the P0 gene by T antigen and c-Jun.

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.

Functional organization of the simian virus 40 origin of DNA replication.

To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T-antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general.

Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen.

We have used two different, but complementary assays to characterize functions of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that several functions were required. These include activities that map to the p53 binding domain and the amino terminal 176 amino acids which contain the J domain as well as the CR1 and CR2 domain required for binding and sequestering the RB family of pocket proteins. Moreover, we found that even though activities dependent only upon the amino terminus were sufficient for immortalization they were unable to maintain it. This suggests that immortalization by these amino terminal functions requires either additional events or immortalization of a subset of cells within the heterogeneous rat embryo fibroblast population. We further found that an activity dependent upon amino acids 17 - 27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions required initially was not required to maintain the immortal state. This represents the first demonstration that some of the functions required for maintenance of the immortal state differ from those required for initiation of immortalization.

Revised sequence of the tetracycline-resistance gene of pBR322.

A revised sequence of the tetracycline-resistance gene of pBR322 is reported. The change, the presence of an additional CG base pair at position 526, adjusts the published sequence to allow an open reading frame from nucleotides 86-1273 (new number) and increases the size of the plasmid to 4363 bp. The predicted polypeptide encoded by this region would contain 396 amino acid residues and have a calculated Mr of 41518. A polypeptide of the predicted size has been reported previously when pBR322 is used as template in the maxicell system.

Instability of HIV sequences in high copy number plasmids.

Plasmid proviral molecular clones of the LAI isolate of HIV-1 and the ROD isolate of HIV-2 were originally prepared in moderate copy number plasmids derived from pBR322, which contains the colE1 origin of replication. In these plasmid vectors, the HIV sequences are stable to continuous passage in bacteria. However, when the colE1 origin was replaced by the mutant origin from pUC18, pGEM, or pBluescript plasmids, which replicates to much higher copy numbers in bacteria, then deletions of HIV sequences occurred even in RecA defective strains. Deletions occurred in two different media, at room temperature and 37 degrees C, and with or without plasmid amplification in the presence of chloramphenicol. These results raise a cautionary note when cloning immunodeficiency viral sequences into plasmid vectors containing a high copy number origin of replication.

Human immunodeficiency virus type 1 infection of antigen-specific CD4 cytotoxic T lymphocytes.

The effect of macrophage (M)-tropic and T cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) infection on antigen-specific CD4 cytotoxic T lymphocytes (CTLs) has been studied using a CD4 CTL line specific for a peptide from influenza B virus hemagglutinin. In the absence of antigen presentation, the production of CC chemokines was low. Both the M-tropic HIV-1 strain (HIV-1AD) and the T-tropic HIV-1 strain (HIV-1LAI) established productive infections in the CD4 CTLs, decreasing antigen-specific cytotoxicity. Peptide presented to the CD4 CTLs increased their secretion of RANTES and MIP-1beta, suppressed M-tropic HIV-1 replication, downmodulated CCR5 expression, and preserved CTL recognition. The suppression of M-tropic HIV-1 replication and downmodulation of the CCR5 receptor likely resulted from CC chemokine secretion since antibodies to CC chemokines restored M-tropic HIV-1 replication. Antigen presentation did not protect CD4 CTLs from T-tropic HIV-1 infection or preserve their CTL recognition. Thus, these CD4 CTLs do not make suppressor factors that inhibit the T-tropic HIV-1LAI isolate. The results indicate that these CD4 CTLs can either harbor or suppress M-tropic HIV-1 infection, depending on whether antigen is present. CD4 CTLs might therefore provide some protection in the early stages of HIV-1 infection when M-tropic isolates are present.

Metallothionein-I promoter-directed expression of foreign proteins in a mouse pituitary corticotrope tumor cell line.

Expressing foreign proteins in heterologous eukaryotic cells has been a powerful tool for analyzing protein structure and function. The inducible mouse metallothionein-I promoter has been particularly useful for expression studies. However, the levels of expression achieved with this promoter in heterologous eukaryotic expression systems have not equaled those observed in vivo for the metallothionein-I gene. We have constructed expression plasmids placing either the gene for chloramphenicol acetyltransferase (CAT) or the cDNA for human neuropeptide Y (NPY) under control of the mouse metallothionein-I promoter. These two expression vectors were used to transfect mouse anterior pituitary tumor cells, from which stable transformants were isolated. The resulting cell lines, Mt.NPY1a and Mt.CAT, were used to maximize functional product expression from the metallothionein-I promoter. In both cell lines, a 35-fold induction of mRNA accumulation, peptide synthesis, or CAT activity was observed.

Analysis of a coded panel of licensed vaccines by polymerase chain reaction-based reverse transcriptase assays: a collaborative study [seecomments].

BACKGROUND: A recent publication reporting the presence of low levels of reverse transcriptase (RT) activity in certain vaccines for human use necessitated that regulatory agencies address the issue of whether this RT activity presented a risk to humans. Detection of low levels of RT activity corresponding to fewer than ten virions became possible with the development of highly-sensitive polymerase chain reaction (PCR)-based RT (PBRT) assays. Variations of the PBRT assay were developed in three laboratories. These assays were reported as being at least one million-fold more sensitive than conventional RT assays. OBJECTIVE: To ascertain the sensitivity and reliability of PBRT assays in different laboratories and to determine which vaccine samples possessed RT activity. STUDY DESIGN: Coded panels of licensed vaccines together with positive and negative controls was assembled at the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration (FDA) and distributed to five cooperating laboratories as well as to our laboratory at CBER. Each laboratory carried out their version of the PBRT assay and submitted the results to the coordinator at CBER. RESULTS: Results of the PBRT analyses carried out in the six laboratories are presented. Five of the six laboratories reported results that were highly consistent. RT activity was detected in live attenuated vaccines that were prepared in chick embryo cells (mumps, measles and yellow fever), but very low or undetectable RT activity was found in vaccines produced in mammalian cells (rabies and rubella). Influenza vaccines from several manufacturers included in the panel displayed the most variability, with different products of this inactivated vaccine having differing amounts of RT activity. CONCLUSIONS: Only vaccines produced in chick embryo cells had significant RT activity. Because RT activity was present in the allantoic fluid of uninfected chick embryos and culture medium from chick embryo fibroblasts, the RT activity arises from the cell substrate used for vaccine production. The PBRT assays were reliably able to detect the low levels of RT activity in chicken-derived vaccines.

The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus.

BACKGROUND: Reverse transcriptase (RT) activity has previously been reported in concentrated medium of primary chicken embryo cell cultures using the traditional RT assay. Recently, using the newly-developed and highly-sensitive product-enhanced reverse transcriptase (PERT) assay, RT activity has been detected in live, attenuated vaccines grown in chicken cell substrates. Furthermore, this activity has been associated with particles that contain RNA related to an ancient, endogenous avian retrovirus family designated as EAV-0. OBJECTIVE: To investigate whether the RT activity present in vaccines produced in specific pathogen-free chicken cell substrates is associated with an infectious retrovirus that can replicate in human cells. STUDY DESIGN: The kinetics of RT activity produced by 10-day-old chicken embryo fibroblast (CEF) cultures was determined by analyzing cell-free medium in a PCR-based RT (PBRT) assay. Material containing the peak PBRT activity was used as the inoculum to infect various human cell lines and peripheral blood mononuclear cells. Filtered supernatants from control and test cultures were analyzed for the presence of replication-competent retroviruses by the PBRT assay. The cells were monitored for other adventitious agents by routine observation for cytopathic effect (CPE) and by transmission electron microscopy (TEM) at culture termination. RESULTS: The PBRT activity did not increase above the background level in the human target cells through at least five cell passages, thus indicating the absence of a replicating retrovirus. No other adventitious agents were detected based upon TEM analysis and the absence of CPE. CONCLUSION: The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.

Production of Schwann cell lines using a regulated oncogene.

The process of myelination in the central and peripheral nervous systems has been well characterized morphologically by a variety of techniques. It is evident from these studies that, in the peripheral nervous system myelin formation is a multistep process. Clearly, a 1:1 relationship must be established with the axon, which is followed by formation of the basal lamina and eventually myelin. Because immortalized Schwann cell lines obtained using SV40 T antigen under the control of an inducible promoter have many properties of untransfected Schwann cells in culture, including their ability to form myelin in vitro, these cells will enable us to dissect more easily the process of myelination. Having successfully immortalized rat Schwann cells without affecting their ability to differentiate fully, we are applying this approach to generate analogous cell lines from the peripheral nerves of other species such as mouse and human. Unlike rat Schwann cells, there are no known mitogens for human and mouse Schwann cells, making it impossible to expand these cell populations. The ability to produce large numbers of human Schwann cells from nerve biopsy and to analyze their biochemical properties would be of enormous value in identifying the cellular abnormalities that result in demyelinating disease. Likewise, there are several mutant mouse strains with defects in myelin formation, and cell lines from these animals would facilitate our understanding of the process leading to dysmyelination.

Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage.

Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells. DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids. Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4-6 copies), and multimerization. Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors. The recombination frequency (NeoR/AmpR) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly. Selective damage in one of the two neo genes increased the relative frequency of gene conversion. The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells.

Complete nucleotide sequence of polyomavirus SA12.

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.