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.

CCR9A and CCR9B: two receptors for the chemokine CCL25/TECK/Ck beta-15 that differ in their sensitivities to ligand.

We isolated cDNAs for a chemokine receptor-related protein having the database designation GPR-9-6. Two classes of cDNAs were identified from mRNAs that arose by alternative splicing and that encode receptors that we refer to as CCR9A and CCR9B. CCR9A is predicted to contain 12 additional amino acids at its N terminus as compared with CCR9B. Cells transfected with cDNAs for CCR9A and CCR9B responded to the chemokine CC chemokine ligand 25 (CCL25)/thymus-expressed chemokine (TECK)/chemokine beta-15 (CK beta-15) in assays for both calcium flux and chemotaxis. No other chemokines tested produced responses specific for the cDNA-transfected cells. mRNA for CCR9A/B is expressed predominantly in the thymus, coincident with the expression of CCL25, and highest expression for CCR9A/B among thymocyte subsets was found in CD4+CD8+ cells. mRNAs encoding the A and B forms of the receptor were expressed at a ratio of approximately 10:1 in immortalized T cell lines, in PBMC, and in diverse populations of thymocytes. The EC50 of CCL25 for CCR9A was lower than that for CCR9B, and CCR9A was desensitized by doses of CCL25 that failed to silence CCR9B. CCR9 is the first example of a chemokine receptor in which alternative mRNA splicing leads to proteins of differing activities, providing a mechanism for extending the range of concentrations over which a cell can respond to increments in the concentration of ligand. The study of CCR9A and CCR9B should enhance our understanding of the role of the chemokine system in T cell biology, particularly during the stages of thymocyte development.

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.

Detection of human immunodeficiency virus type 1 after infection of unstimulated peripheral blood mononuclear cells.

Application of a highly sensitive PCR-based reverse transcriptase (RT) assay to the analysis of the infection of CD4+ cell lines with human immunodeficiency virus type 1 (HIV-1) demonstrated that virus production can be detected as early as 24 h after infection. Most of the signal at 24 h was due to virus production, as it could be substantially reduced by prior treatment with the RT inhibitor zidovudine. Virus production at 24 and 48 h was unaffected by the protease inhibitor indinavir. Infection of unstimulated peripheral blood mononuclear cells (PBMC) with a macrophage-tropic HIV-1 isolate yielded increasing virus production for 2-3 weeks, while infection with a T-cell line-tropic isolate yielded only low and sporadic virus production. Productive infection of unstimulated PBMC by the macrophage-tropic virus required functional Gag matrix and Vpr proteins; therefore, the monocyte-derived macrophage is probably the virus-producing cell in these cultures.

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.

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.

CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection.

Thymocyte infection with HIV-1 is associated with thymic involution and impaired thymopoiesis, particularly in pediatric patients. To define mechanisms of thymocyte infection, we examined human thymocytes for expression and function of CXCR4 and CCR5, the major cell entry coreceptors for T cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) strains of HIV-1, respectively. CXCR4 was detected on the surface of all thymocytes. CXCR4 expression on mature, high level TCR thymocytes was similar to that on peripheral blood T cells, but was much lower than that on immature thymocytes, including CD34+ thymic progenitors. Consistent with this, stroma-derived factor-1 (SDF-1) induced calcium flux primarily in immature thymocytes, with CD34+ progenitors giving the strongest response. In addition, SDF-1 mRNA was detected in thymic-derived stromal cells, and SDF-1 induced chemotaxis of thymocytes, suggesting that CXCR4 may play a role in thymocyte migration. Infection of immature thymocytes by the T-tropic HIV-1 strain LAI was 10-fold more efficient than that in mature thymocytes, consistent with their relative CXCR4 surface expression. Anti-CXCR4 antiserum or SDF-1 blocked fusion of thymocytes with cells expressing the LAI envelope. In contrast to CXCR4, CCR5 was detected at low levels on thymocytes, and CCR5 agonists did not induce calcium flux or chemotaxis in thymocytes. However, CD4+ mature thymocytes were productively infected with the CCR5-tropic strain Ba-L, and this infection was specifically inhibited with the CCR5 agonist, macrophage inflammatory protein-1beta. Our data provide strong evidence that CXCR4 and CCR5 function as coreceptors for HIV-1 infection of human thymocytes.

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission.

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.

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.

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP.

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.

Maintenance of an unusual polypurine tract in HIV-2: stability to passage in culture.

A stretch of purine residues, the polypurine tract (PPT), is found in all retroviruses and is used to initiate plus-strand DNA synthesis. While the PPT of most lentiviruses is a homogeneous sequence of purine residues, the PPT of some isolates of the human and simian immunodeficiency viruses is interrupted with a single pyrimidine residue. The ROD strain of human immunodeficiency virus type 2 (HIV-2) has such a pyrimidine-containing variant PPT. Virus generated from an infectious molecular clone, pROD10, was used to infect two CD4-positive T-cell lines, H9 and CEM. The sequence of the PPT was determined after two passages. From both cell lines, the variant PPT was retained, demonstrating that the presence of a pyrimidine in the PPT was fully functional and that there was no strong selection for an all-purine PPT.

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.

Both virus and host components are important for the manifestation of a Nef- phenotype in HIV-1 and HIV-2.

While it has been demonstrated that the Nef protein of simian immunodeficiency virus is obligatory for the establishment of high viral loads and the development of simian AIDS in rhesus macaques, demonstrating a critical role for the human immunodeficiency virus (HIV) Nef protein in tissue culture has been elusive. Data have been contradictory as to whether Nef has a negative or positive influence on in vitro virus replication. In an attempt to define a role for Nef during virus propagation in tissue culture and to obtain virus-host systems that could distinguish between the Nef mutant and wild-type viruses, we have introduced mutations into the nef genes of infectious molecular clones of three HIV-1 strains and two isolates of the HIV-2ROD strain and have investigated the capacity of viruses derived from them to infect a number of CD4-positive T-cell lines and peripheral blood mononuclear cells (PBMC). Mutating the nef gene of all viruses had a modest negative effect on virus production in activated PBMC. In some T-cell lines with some viruses, the effects were severe, and little or no Nef mutant virus could be detected. In other cell lines, the result of mutating the nef gene either had no effect or had a slight negative effect on the replication kinetics. Therefore, whether the consequences of loss of Nef activity can be demonstrated in vitro depends on both the particular virus and the host cell used, suggesting that Nef is exerting its activity on some cellular pathway. In addition, we describe the construction and properties of hitherto unreported infectious molecular clones of the ROD strain of HIV-2.

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.