Eugene V. Barsov

Proteomic and Biochemical Analysis of Purified Human Immunodeficiency Virus Type 1 Produced from Infected Monocyte-Derived Macrophages

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects CD4+ T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

The structural complexity of the human BORIS gene in gametogenesis and cancer.

Background BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions. Methodology/Principal Findings Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates. Conclusions The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.

Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

ABSTRACT We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing theenv-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 × 106 CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

Transduction of SIV-specific TCR genes into rhesus macaque CD8+ T cells conveys the ability to suppress SIV replication.

Background The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8+ T-cell control of SIV replication in CD4+ T cells, we asked whether TCRs isolated from rhesus macaque CD8+ T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8+ T cells obtained from an uninfected/unvaccinated animal. Principal Findings We transferred SIV-specific TCR genes isolated from rhesus macaque CD8+ T-cell clones with varying abilities to suppress SIV replication in vitro into CD8+ T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8+ T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones. Conclusions Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8+ T cells independent of IFN-γ and CD107a responses

CD8(+) T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8(+) T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4(+) T cells. We used a set of SIV(mac)239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I(high) or MHC class I(intermediate). However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I(low)) despite the observations that all CTL clones showed similar IFN-gamma responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.

The Mamu B*17-restricted SIV Nef IW9 to TW9 mutation abrogates correct epitope processing and presentation without loss of replicative fitness

CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.

Selective immortalization of tumor-specific T cells to establish long-term T-cell lines maintaining primary cell characteristics.

Antigen-specific T cells play a key role in cellular immune response against cancer. The ability to isolate, maintain, and characterize tumor-specific T cells is a prerequisite to studying anticancer immune response and developing novel strategies for cancer immunotherapy. However, the life span of human T cells in vitro is usually short and is limited by the onset of cellular senescence. To establish long-term, antigen-specific T-cell lines and clones, we selectively immortalized antigen-responsive T cells from human peripheral blood mononuclear cells (PBMCs). PBMCs were stimulated with antigens, and then infected with a murine leukemia virus-based retroviral vector carrying an immortalizing gene, the human telomerase-reverse transcriptase gene. Since such vectors can only integrate in dividing cells, only antigen-activated T cells are efficiently transduced. Using this approach, we generated immortalized T-cell lines that maintained strictly IL-2-dependent growth and MHC-restricted, antigen-specific responsiveness, some of which have been in continuous culture for longer than 1 year, far in excess of the survival of parallel control nonimmortalized cultures. These lines showed antigen-specific proliferation with induced cytokine and chemokine production, and, in the case of CD8+ T-cell lines, antigen-specific cytolytic activity. When applied to the tumor antigen-specific T cells, the approach provides a convenient, reproducible means for generating a stable, continuously renewable source of antigen-specific T lymphocytes for a variety of studies on anticancer immunity.

TCR triggering transcriptionally downregulates CCR5 expression on rhesus macaque CD4(+) T-cells with no measurable effect on susceptibility to SIV infection.

Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4(+) T-cell clones revealed marked differences in susceptibility to SIV(mac)239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIV(mac)239 susceptibility. Macaque CD4(+) T-cells showed significant CCR5 downregulation 1-2days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10days after activation. Exposure of clones to SIV(mac)239 during their CCR5(low) or CCR5(high) expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIV(mac)239 infection of clones during their CCR5(low) or CCR5(high) expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.

Mutations of a residue within the polyproline-rich region of Env alter the replication rate and level of cytopathic effects in chimeric avian retroviral vectors.

ABSTRACT Previous attempts to extend the host range of the avian sarcoma/leukosis virus (ASLV)-based RCASBP vectors produced two viral vectors, RCASBP M2C (4070A) and RCASBP M2C (797-8), which replicate using the amphotropic murine leukemia virus 4070A Env protein (2). Both viruses were adapted to replicate efficiently in the avian cell line DF-1, but RCASBP M2C (4070A) caused extensive cytopathic effects (CPE) in DF-1 cells whereas RCASBP M2C (797-8) induced low levels of CPE. The two viruses differed only at amino acid 242 of the polyproline-rich region in the surface (SU) subunit of the Env protein. In RCASBP M2C (4070A), an isoleucine replaced the wild-type proline residue, whereas a threonine residue was found in RCASBP M2C (797-8). In the present study, we show that other amino acid substitutions at position 242 strongly influence the CPE and replication rate of the chimeric viruses. There was a correlation between the amount of unintegrated linear retroviral DNA present in infected DF-1 cells and the level of CPE. This suggests that there may be a role for superinfection in the CPE. The treatment of RCASBP M2C (4070A)-infected cells with dantrolene, which inhibits the release of calcium from the endoplasmic reticulum (ER), reduced the amount of CPE seen during infection with the highly cytotoxic virus. Dantrolene treatment did not appear to affect virus production, suggesting that Ca2+ release from the ER had a role in the CPE caused by these viruses.