Peter R. Shank

Novel Diversity in IL-4-Mediated Responses in Resting Human Naive B Cells Versus Germinal Center/Memory B Cells

Recent studies have defined several phenotypic and molecular changes associated with the maturation of naive human B cells within the milieu of germinal centers. Although naive B cells serve as natural precursors to germinal center (GC)/memory (M) subpopulations, little is known about the physiological requirements for the survival of the naive B cell pool in the absence of cell-cell contact or Ag-mediated activation. Because IL-4 induces expression of several membrane receptors such as CD23 which are uniquely present on resting human naive B lymphocytes, we hypothesized that these cells might be intrinsically programmed to respond to IL-4 in the absence of cell division. Using buoyant density-dependent isolation and further enrichment by negative/positive selection of human naive and GC/M subpopulations, we characterized cytokine receptor moieties on these cells and analyzed their survival and growth in the presence of IL-4 or IL-10. Resting naive B cells expressed significantly higher IL-4 receptor α-chain on their cell surface than the combined GC/M subpopulation. The IL-10 receptor and the IL-2 receptor γc chain were almost equally expressed on both subpopulations. When cultured in vitro, the addition of IL-4, but not IL-10, protected naive B cells from apoptosis in the absence of activation and growth. However, IL-4 exerted no such effect on resting GC/M B cells. These data support the hypothesis that IL-4 plays a pivotal role in the survival and maintenance of resting human naive B cells.

CD4-Independent infection of human B cells with hiv type 1: Detection of unintegrated viral DNA

Although B lymphocytes are a major constituent of lymphoid organs and acquire a significantly altered phenotype and function in HIV-infected individuals, it remains unclear whether CD4-negative B cells are a susceptible host for viral entry and long-term productive infection. We screened a number of Epstein-Barr virus (EBV)-positive and-negative Burkitts lymphoma (BL) B cell lines as well as subpopulations of normal B cells that include tonsillar naive and germinal center/memory B cells for the expression of HIV-1 receptors CD4, CXCR4, and CCR5. Cell lines and resting or activated normal B cells lacked CD4 and CCR5 but expressed CXCR4. We demonstrate HIV-1 infection of a CD4-negative, EBV-negative (BL) cell line, CA46, which remained productively infected yet noncytopathic for more than 36 months in culture. HIV-1 (HTLV-III(B)) infection of CA46 cells was mediated through CXCR4 in a CD4-independent manner and correlated with upregulation of the expression of B cell activation markers CD23 and CD95 (Fas receptor). Despite Fas receptor expression, HIV-1-infected CA46 cells remained resistant to Fas-mediated cell death. CA46-derived, CD4-independent viral isolates were proficient in infecting and causing syncytium formation in Molt4 T cells. The HIV-1 genomic organization in persistently infected CA46 clones was found to be predominantly unintegrated linear and circular DNA. Importantly, naive and germinal center/memory B cells could also be infected by HIV-1 in a CD4-independent manner. Although these B cell subpopulations expressed moderate to high levels of CXCR4, they required activation through CD40 and interleukin 4 receptor for infection. These findings point to B cells as an additional HIV-1 target and suggest a structural evolution of the HIV-1 genome responsible for CD4-independent and noncytopathic infections.

γ-Tubulin Overexpression in Sertoli Cells In Vivo. II: Retention of Spermatids, Residual Bodies, and Germ Cell Apoptosis

Abstract The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, γ-tubulin, were delivered to Sertoli cells in vivo. γ-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.

γ-Tubulin Overexpression in Sertoli Cells In Vivo: I. Localization to Sites of Spermatid Head Attachment and Alterations in Sertoli Cell Microtubule Distribution

Abstract Sertoli cells play a number of roles in supporting spermatogenesis, including structural organization, physical and paracrine support of germ cells, and secretion of factors necessary for germ cell development. Studies with microtubule disrupting compounds indicate that intact microtubule networks are crucial for normal spermatogenesis. However, treatment with toxicants and pharmacologic agents that target microtubules lack cell-type selectivity and may therefore elicit direct effects on germ cells, which also require microtubule-mediated activities for division and morphological transformation. To evaluate the importance of Sertoli cell microtubule-based activities for spermatogenesis, an adenoviral vector that overexpresses the microtubule nucleating protein, γ-tubulin, was used to selectively disrupt microtubule networks in Sertoli cells in vivo. γ-Tubulin overexpression was observed to cause redistribution of Sertoli cell microtubule networks, and overexpression of a γ-tubulin-enhanced green fluorescent protein fusion protein was observed to localize to the site of elongate spermatid head attachment to the seminiferous epithelium.

Restriction endonuclease mapping of the DNA of Rous-Associated virus 0 reveals extensive homology in structure and sequence with avian sarcoma virus DNA

Abstract We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells.PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA (PvuI andEcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 106Mr and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[ Shank, P. R.,et al. (1978b) .Cell15, 1383–1395; Hsu, W.,et al. (1978) .J. Virol.28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.

Analysis of Proto-oncogene Expression During Liver Regeneration and Hepatocarcinogenesis

The discovery and characterization of viral oncogenes and the cellular genes from which they originated (cellular or proto-oncogenes) represents one of the most dramatic advances in the field of cancer research in recent years. Elucidation of the role of these genes in the process of oncogenesis and the role of the proto-oncogenes in the control of normal cellular growth and/or differentiation has united the fields of cancer research and cell biology. Despite the vast amount of information which has become available recently on the molecular nature of the alterations which lead to the “activation” of protooncogenes, the precise role of these genes in the development of natural tumors remains to be established. Some investigators have gone so far as to state that “there is as yet no convincing evidence that activated proto-oncogenes are even necessary, much less sufficient for carcinogenesis” [1]. Although this rather extreme view is a minority opinion, it is important to remember that in the complex process encompassing the development of a tumor, the oncogene may be but one player with several others remaining to be elucidated. Given this still unsettled picture, it is particularly important to define the role which various proto-oncogenes play in normal cells and tissues.

Molecular analysis of episomal human papillomavirus type 16 DNA in a cervical carcinoma cell line

Integration of human papillomavirus type 16 DNA sequences into host DNA is a frequent event in cervical carcinogenesis. However, recent studies showing that HPV16 is present exclusively in an episomal form in many primary cervical cancers suggest that HPV16 can transform target cells by mechanisms that do not require viral integration. We have established a cervical carcinoma cell line that harbors episomal copies of HPV16 DNA of approximately 10 kb. Restriction enzyme and two-dimensional gel analysis confirmed that HPV16 DNA was extrachromosomal with both monomeric and multimeric forms present. HPV16 was maintained as episomes with passage both in culture and after subcutaneous growth in nude mice. The 10 kb viral genome, consisting of a full-length copy of HPV16 and a partial duplication of the long control region and the L1 open reading frame, exhibited transforming activity comparable to prototype HPV16. This cell line should provide a useful model system for studying the biological significance of the physical state of the HPV16 genome in cervical carcinoma cells.

Identification and characterization of dimeric and trimeric circular forms of avian sarcoma virus-specific DNA

Abstract Covalently closed circular forms of viral DNA are found in the nuclei of cells acutely infected by avian sarcoma virus. In this report we show that dimeric and trimeric forms of closed circular DNA are present, in addition to the previously characterized, predominant monomeric species. The identities of these oligomeric circular molecules were verified by their characteristic behavior during equilibrium centrifugation in cesium chloride-propidium diiodide gradients, rate-zonal centrifugation in sucrose gradients, and electrophoresis in agarose gels, before and after digestion with nucleases. Restriction endonuclease cleavage of isolated dimeric and trimeric molecules indicates that they consist of monomers linked in a “head-to-tail” orientation.

Nucleotide sequence comparison of the 3′ regions of avian retroviruses NY203 and NTRE-2

We have been characterizing molecular clones of two subgroup E avian retroviruses (NTRE-2 and NY203RAV-60) that produce different proliferative diseases after inoculation into susceptable K28 chickens. Both viruses arose by recombination between exogenous and endogenous viral genomes. To further characterize regions of these viruses that are important for the production of disease, we have determined the nucleotide sequence of a 1.2-kb EcoRI fragment extending from the carboxyl end of gp85 through 150 bases of the U3 region of the LTR. From the sequence data it is possible to precisely define one point where recombination occurred between PrRSV-B and RAV-0 to produce NTRE-2. We suggest a hypothesis, based on the core enhancer consensus sequence, for the higher incidence of disease when chickens are infected with viruses bearing the LTR of NY203RAV-60.

Tetranucleotide usage highlights genomic heterogeneity among mycobacteriophages.

Background The genomic sequences of mycobacteriophages, phages infecting mycobacterial hosts, are diverse and mosaic. Mycobacteriophages often share little nucleotide similarity, but most of them have been grouped into lettered clusters and further into subclusters. Traditionally, mycobacteriophage genomes are analyzed based on sequence alignment or knowledge of gene content. However, these approaches are computationally expensive and can be ineffective for significantly diverged sequences. As an alternative to alignment-based genome analysis, we evaluated tetranucleotide usage in mycobacteriophage genomes. These methods make it easier to characterize features of the mycobacteriophage population at many scales. Description We computed tetranucleotide usage deviation (TUD), the ratio of observed counts of 4-mers in a genome to the expected count under a null model. TUD values are comparable between members of a phage subcluster and distinct between subclusters. With few exceptions, neighbor joining phylogenetic trees and hierarchical clustering dendrograms constructed using TUD values place phages in a monophyletic clade with members of the same subcluster. Regions in a genome with exceptional TUD values can point to interesting features of genomic architecture. Finally, we found that subcluster B3 mycobacteriophages contain significantly overrepresented 4-mers and 6-mers that are atypical of phage genomes. Conclusions Statistics based on tetranucleotide usage support established clustering of mycobacteriophages and can uncover interesting relationships within and between sequenced phage genomes. These methods are efficient to compute and do not require sequence alignment or knowledge of gene content. The code to download mycobacteriophage genome sequences and reproduce our analysis is freely available at https://github.com/bsiranosian/tango_final.