Jason D. Walker

SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of ∼16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

Evidence that acetyl phosphate functions as a global signal during biofilm development

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüß and Wolfe, 1994, Mol Microbiol 12: 973–984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild‐type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co‐ordinate the expression of surface structures and cellular processes involved in the initial stages of wild‐type biofilm development.

Cytomegalovirus-Infected Human Endothelial Cells Can Stimulate Allogeneic CD4+ Memory T Cells by Releasing Antigenic Exosomes

Human CMV infection is controlled by T cell-mediated immunity and in immunosuppressed transplant patients it is associated with acute allograft rejection as well as chronic allograft vasculopathy. CMV infects endothelial cells (EC) and it is thought that CMV-specific host immune responses to infected allograft EC contribute to rejection. In vitro, CD4+ T cells from CMV-positive donors (but not CMV-negative donors) are readily activated by CMV-infected allogeneic EC, although it is unclear how allogeneic CMV-infected EC activate self-class II MHC-restricted memory CD4+ T cells. In this study, we confirm that purified CD4+ T cells from CMV+ donors are activated by allogeneic CMV-infected EC, but find that the response is dependent upon copurified APC expressing class II MHC that are autologous to the T cells. The transfer of CMV Ags from infected EC to APC can be mediated by EC-derived exosome-like particles. These results provide a mechanism by which CMV can exacerbate allograft rejection and suggest a novel function of EC-derived exosomes that could contribute in a more general manner to immune surveillance.

SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes.

Endoreplication, also known as endoreduplication, is a phyogenetically widespread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas overexpression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.

Functional Hierarchy of Herpes Simplex Virus 1 Viral Glycoproteins in Cytoplasmic Virion Envelopment and Egress

ABSTRACT Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115–6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2, engineered not to express either UL11 or gM, replicated more than 1 log unit less efficiently and produced significantly smaller plaques than UL11-null or gM-null viruses alone, in agreement with the results of Leege et al. (T. Leege et al., J. Virol. 83:896-907, 2009). Analyses of particle-to-PFU ratios, relative plaque size, and kinetics of virus growth and ultrastructural visualization of glycoprotein-deficient mutant and wild-type virions indicate that gDΔct, gE, and gM function in a cooperative but not redundant manner in infectious virion morphogenesis. Overall, comparisons of single, double, and triple mutant viruses generated in the same HSV-1(F) genetic background indicated that lack of either UL20 or gK expression caused the most severe defects in cytoplasmic envelopment, egress, and infectious virus production, followed by the double deletion of UL11 and gM.

Oncolytic herpes simplex virus 1 encoding 15-prostaglandin dehydrogenase mitigates immune suppression and reduces ectopic primary and metastatic breast cancer in mice.

ABSTRACT Oncolytic herpes simplex virus 1 (HSV-1) viruses armed with immunomodulatory transgenes have shown potential for enhanced antitumor therapy by overcoming tumor-based immune suppression and promoting antitumor effector cell development. Previously, we reported that the new oncolytic HSV-1 virus, OncSyn (OS), engineered to fuse tumor cells, prevented tumor growth and metastasis to distal organs in the 4T1/BALB/c immunocompetent breast cancer mouse model, suggesting the elicitation of antitumor immune responses (Israyelyan et al., Hum. Gen. Ther. 18:5, 2007, and Israyelyan et al., Virol. J. 5:68, 2008). The OSV virus was constructed by deleting the OS viral host shutoff gene (vhs; UL41) to further attenuate the virus and permit dendritic cell activation and antigen presentation. Subsequently, the OSVP virus was constructed by inserting into the OSV viral genome a murine 15-prostaglandin dehydrogenase (15-PGDH) expression cassette, designed to constitutively express 15-PGDH upon infection. 15-PGDH is a tumor suppressor protein and the primary enzyme responsible for the degradation of prostaglandin E2 (PGE2), which is known to promote tumor development. OSVP, OSV, and OS treatment of 4T1 tumors in BALB/c mice effectively reduced primary tumor growth and inhibited metastatic development of secondary tumors. OSVP was able to significantly inhibit the development and accumulation of 4T1 metastatic tumor cells in the lungs of treated mice. Ex vivo analysis of immune cells following treatment showed increased inflammatory cytokine production and the presence of mature dendritic cells for the OSVP, OSV, and OS viruses. A statistically significant decrease in splenic myeloid-derived suppressor cells (MDSC) was observed only for OSVP-treated mice. These results show that intratumoral oncolytic herpes is highly immunogenic and suggest that 15-PGDH expression by OSVP enhanced the antitumor immune response initiated by viral infection of primary tumor cells, leading to reduced development of pulmonary metastases. The availability of the OSVP genome as a bacterial artificial chromosome allows for the rapid insertion of additional immunomodulatory genes that could further assist in the induction of potent antitumor immune responses against primary and metastatic tumors.

Site-Specific Proteolytic Cleavage of the Amino Terminus of Herpes Simplex Virus Glycoprotein K on Virion Particles Inhibits Virus Entry

ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431–12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310–6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.

Endothelial Cells Promote Human Immunodeficiency Virus Replication in Nondividing Memory T Cells via Nef-, Vpr-, and T-Cell Receptor-Dependent Activation of NFAT

ABSTRACT Human endothelial cells (ECs) enhance human immunodeficiency virus (HIV) replication within CD4+ memory T cells by 50,000-fold in a Nef-dependent manner. Here, we report that EC-mediated HIV type 1 replication is also dependent on an intact vpr gene. Moreover, we demonstrate that despite a requirement for engaging major histocompatibility complex (MHC) class II molecules and costimulators, EC-stimulated virus-producing cells (p24high T cells) do not proliferate, nor are they arrested in the cell cycle. Rather, they are minimally activated, sometimes expressing CD69 but not CD25, HLA-DR, VLA-1, or effector cytokines. Blocking antibodies to interleukin 2 (IL-2), IL-6, IL-7, or tumor necrosis factor do not inhibit viral replication. Cyclosporine effectively inhibits viral replication, as does disruption of the NFAT binding site in the viral long terminal repeat. Furthermore, in the presence of ECs, suboptimal T-cell receptor (TCR) stimulation with phytohemagglutinin L supports efficient viral replication, and suboptimal stimulation with toxic shock syndrome toxin 1 leads to viral replication selectively in the TCR-stimulated, Vβ2-expressing T cells. Collectively, these data indicate that ECs provide signals that promote Nef- and Vpr-dependent HIV replication in memory T cells that have been minimally activated through their TCRs. Our studies suggest a mechanism for HIV replication in vivo within the reservoir of circulating memory CD4+ T cells that persist despite antiretroviral therapy and further suggest that maintenance of immunological memory by MHC class II-expressing ECs via TCR signaling may contribute to HIV rebound following cessation of antiretroviral therapy.

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

ABSTRACT Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.

Human Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in CD4+ T Cells in a Nef-Dependent Manner In Vitro and In Vivo

ABSTRACT Infected CD4+ T cells are the primary sites of human immunodeficiency virus type 1 (HIV-1) replication in vivo. However, signals from professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, greatly enhance HIV-1 replication in T cells. Here, we report that in cocultures, vascular endothelial cells (ECs), which in humans can also serve as APCs, can enhance HIV-1 production of both CCR5- and CXCR4-utilizing strains approximately 50,000-fold. The observed HIV-1 replication enhancement conferred by ECs occurred only in memory CD4+ T cells, required expression of major histocompatibility complex class II (MHC-II) molecules by the ECs, and could not be conferred by fixed ECs, all of which are consistent with a requirement for EC-mediated T-cell activation via T-cell receptor (TCR) signaling. Deletion of nef (Nef−) decreased HIV-1 production by approximately 100-fold in T cells cocultured with ECs but had no effect on virus production in T cells cocultured with professional APCs or fibroblasts induced to express MHC-II. Human ECs do not express B7 costimulators, but Nef− replication in CD4+-T-cell and EC cocultures could not be rescued by anti-CD28 antibody. ECs act in trans to enhance wild-type but not Nef− replication and facilitate enhanced wild-type replication in naïve T cells when added to T-cell or B-lymphoblastoid cell cocultures, suggesting that ECs also provide a TCR-independent signal to infected T cells. Consistent with these in vitro observations, wild-type HIV-1 replicated 30- to 50-fold more than Nef− in human T cells infiltrating allogeneic human skin grafts on human huPBL-SCID/bg mice, an in vivo model of T-cell activation by ECs. Our studies suggest that ECs, which line the entire cardiovascular system and are, per force, in frequent contact with memory CD4+ T cells, provide signals to HIV-1-infected CD4+ T cells to greatly enhance HIV-1 production in a Nef-dependent manner, a mechanism that could contribute to the development of AIDS.