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PubMed2006

KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo.

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV, purified HPCs were exposed to KSHV, and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells, and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection, persistence, and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo.

Animals↗
PubMed2000

Identification of a domain in human immunodeficiency virus type 1 rev that is required for functional activity and modulates association with subnuclear compartments containing splicing factor SC35.

The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the DeltaLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Myeloid cell kinetics in response to haemopoietic growth factors.

The morphologically recognizable cells of haemopoietic tissue comprise some 95% of the total and their kinetic performance is both flexible and adaptable to stress conditions. The effects of the common myeloid growth factors on these cells have been examined. Using the classically developed analyses of cell kinetics with tritiated thymidine labelling autoradiography, it is shown that continuously infused or repeated injections of G-CSF increases the proliferative activity of marrow haemopoietic tissue in both mouse and man, shortening the average cell cycle times by 35 and 65% respectively, amplifying neutrophil production so that levels in the peripheral blood rise 10-15-fold. This amplified production, amounting to 3-4 extra proliferation divisions, is mostly confined to the proliferatively active maturing neutrophil cell compartments. In addition, the neutrophil maturation time is also reduced, leading to a rapid and sustained release of postmitotic cells within 1-2 days, compared with the normal time of 4-6 days. Neither GM-CSF nor IL-3 generates any significant amplification of neutrophil production in mice but, in humans, GM-CSF stimulates cell proliferation in the bone marrow to a similar degree as doses G-CSF. Studies on the granulocyte-macrophage progenitor population show that the proliferation stimulus in response to GM-CSF is not confined to the maturing populations. In the maturing neutrophil precursor population, average cell cycle times are shortened by 60%, but in this case the overall maturation times are unaffected and their time of release into the circulation is normal. The response to GM-CSF, however, is not so straightforward as that to G-CSF. The peripheral half-life of the mature cells is considerably prolonged and this is consistent with some suggestion of functional impairment. In addition, a significant release of immature cells and eosinophils (also expanded in the bone marrow in response to GM-CSF) dilutes the neutrophilic response. Monocyte production is also stimulated by G- and GM-CSF and, though no direct measurement of proliferation has been made, stimulation at all stages of their proliferation, maturation and release are implied.

Agranulocytosis↗
PubMed2005

Scrambling of the amino acids within the transmembrane domain of Vpu results in a simian-human immunodeficiency virus (SHIVTM) that is less pathogenic for pig-tailed macaques.

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.

Acquired Immunodeficiency Syndrome↗
PubMed1987

DNA synthesis in pulmonary alveolar macrophages and type II cells: effects of ozone exposure and treatment with alpha-difluoromethylornithine.

An increase in the number of pulmonary alveolar macrophages (AM) can be induced by a number of toxic insults to the lung, including ozone, an important photochemical oxidant air pollutant. This increase could arise from an influx of monocytes from the vascular or interstitial compartments, or from proliferation of AM in situ. While proliferation of alveolar type II cells after oxidant exposure has been well documented, it is not clear whether AM are also capable of this response. Rats were exposed to air or to 0.12, 0.25, or 0.50 ppm ozone for 1, 2, 3, 7, or 14 d, 20 h/d. The labeling index in both AM and type II cells increased about 10-fold after 2 d of exposure to 0.25 and 0.50 ppm of ozone, but returned to control levels by the end of 1 wk of exposure. These changes closely paralleled the temporal and dose-response characteristics of changes in total lung DNA synthesis. alpha-Difluoromethylornithine (DFMO) administered to rats during a 2-d exposure to 0.50 ppm ozone did not inhibit the ozone-induced increase in labeling index in AM or type II cells, although evidence of inhibition of lung ornithine decarboxylase activity was obtained, and the ozone-induced increase in total lung DNA synthesis was inhibited by 23%. These results suggest that, like type II cells, AM are capable of entering the cell cycle and synthesizing new DNA in situ in response to short-term exposure to environmentally relevant doses of ozone, and that the ozone-induced stimulation of DNA synthesis in these cell types was refractory to inhibition by DFMO.

Animals↗
PubMed1999

Chemokines and the inflammatory response to viral infection in the central nervous system with a focus on lymphocytic choriomeningitis virus.

Leukocyte migration to the central nervous system (CNS) is a common process with often devastating consequences that follows infection of this tissue compartment with a variety of viruses. The mechanisms underlying this process are poorly defined but, it is hypothesized that chemokines may be important regulatory signals for the cerebral recruitment and extravasation of leukocytes. Here we discuss this hypothesis in the context of different viral infections of the CNS with emphasis on lymphocytic choriomeningitis virus (LCMV). In general, the pattern of chemokine gene expression in these CNS viral infections is dynamic and complex with often overlapping expression of a number of different subclasses of chemokine genes. In the case of CNS infection with LCMV, cerebral chemokine gene expression was observed in euthymic and to a lesser extent athymic mice and preceded increases in cytokine gene expression and in euthymic mice, CNS leukocyte recruitment. These observations together with the finding that CRG-2/IP-10, a prominently expressed chemokine gene in many different CNS viral infections, was expressed by cells intrinsic to the CNS e.g. astrocytes, suggest that activation of chemokine gene expression may be a direct, early and localized host response to viral infection. These findings are consistent with the proposed involvement of chemokines as key signaling molecules for the migration of leukocytes to the CNS following virus infection.

Animals↗
PubMed2000

Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G1 phase of the cell cycle.

Human cytomegalovirus blocks cell-cycle progression in the G(1) compartment upon infection of primary human fibroblasts. The virus-coded UL69 protein can institute a G(1) block when expressed in cells in the absence of virus infection. We have constructed a cytomegalovirus mutant, TNsubUL69, that lacks the UL69 coding region. This virus grows slowly in fibroblasts, but produces a wild-type yield after an extended delay. It grows with normal kinetics in cells coinfected with a recombinant retrovirus, retroUL69, which expresses UL69 protein, demonstrating that its growth defect results from the mutation in the UL69 gene. UL69 protein is packaged within virus particles, and it was possible for us to produce two types of virus stocks. TNsubUL69(+pUL69) lacks the UL69 gene but contains UL69 protein in virus particles. It is produced by growth in fibroblasts that are coinfected with retroUL69. TNsubUL69(-pUL69) lacks the UL69 gene and protein. It is produced by growth in fibroblasts that do not contain UL69 protein. The mutant virions lacking both the UL69 gene and protein fail to induce a cell-cycle block with normal efficiency, whereas the mutant particles lacking the gene but containing the protein can institute the block. These results are consistent with the view that the UL69 protein contributes to the cytomegalovirus-induced cell-cycle block, and they suggest that UL69 protein delivered to cells within virions can induce the block without the synthesis of additional UL69 protein encoded by the infecting viral genome.

Cell Cycle↗
PubMed1988

Prospects for HIV vaccines.

This article initially discusses the types of responses elicited by infectious agents, such as viruses and the role of each response in preventing, limiting, and clearing the infection. An important response is the generation of immunological memory, in both the B and T cell compartments. Generally, attenuated viral vaccines have been highly successful at inducing long-lived immunity but our understanding of the reasons for this comes from the study of model systems, such as murine influenza virus infections. Specific antibody may largely prevent infection and specific cytotoxic T cells and antibody-dependent cell cytotoxic reactions are the main mechanisms for clearing viral infections. Recent evidence shows that for some months after infection by HIV, a strong cytotoxic T (Tc) cell response occurs in infected, asymptomatic individuals; these cells are continuously generated by HIV-infected stimulator cells that most likely also serve as target cells in vivo. A low level of specific antibody is also formed and a number of reasons are listed to explain why HIV escapes antibody-mediated neutralization and infects cells expressing CD4 receptors. Cells of the macrophage/monocyte lineage are also infected and these express Fc and complement receptors; there is the strong possibility that infection of these cells occurred following the formation of complexes of infectious HIV with antibody to the surface antigen and attachment of complement components. The continuous presence of activated Tc cells that, in contrast to many other viral diseases, does not clear the infection strongly suggests that foci of infected cells sequestered from or resistant to immune control become established. These secrete virus that infects other (stimulator) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1990

Virus entry and antigen biosynthesis in the processing and presentation of class-II MHC-restricted T-cell determinants of influenza virus.

Receptor-mediated uptake of influenza virus is responsible for efficient introduction of virus particles to APC. This leads to the effective presentation to T-cells of very small concentrations of proteins entering on the intact virus. Endocytosed virus transits rapidly to the endosome compartment. Entry into this environment appears to greatly affect the fate of T-cell determinants. While promoting the presentation of determinants which require extensive antigen processing, the intracellular environment appears also to lead to destruction of labile determinants, such as those of NA. The same NA determinants are efficiently presented by actively infected cells, indicating that newly biosynthesized viral proteins need not be subjected to the same handling as internalized viral particles. In a similar way, site 3 of HA, which, in a single pulse of noninfectious virus or isolated HA protein is expressed with a relatively short half-life, has greatly improved levels of duration and expression on actively infected APC. Since certain T(H) determinants are unavailable or poorly expressed when introduced on nonreplicative influenza virus, vaccination with inactivated virus might have limitations in stimulating T(H) as well as class-I responses. Finally, individual T-cell determinants of the same protein can exhibit distinct patterns of expression and persistence on APC surfaces. These different half-lives of T(H) determinants may be influential in determining immuno-dominance of T-cell sites. Determinants that are longer-lived on APC may have a greater probability of interacting with appropriate T(H) precursors, which could lead to an enhanced T-cell response to that region of the viral protein.

Animals↗
PubMed2002

Addition of exogenous protease facilitates reovirus infection in many restrictive cells.

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.

Animals↗
PubMed2005

Compartmentalized human immunodeficiency virus type 1 present in cerebrospinal fluid is produced by short-lived cells.

Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) during primary infection and persists in this compartment by unknown mechanisms over the course of infection. In this study, we examined viral population dynamics in four asymptomatic subjects commencing antiretroviral therapy to characterize cellular sources of HIV-1 in the CNS. The inability to monitor viruses directly in the brain poses a major challenge in studying HIV-1 dynamics in the CNS. Studies of HIV-1 in cerebrospinal fluid (CSF) provide a useful surrogate for the sampling of virus in the CNS, but they are complicated by the fact that infected cells in local CNS tissues and in the periphery contribute to the population pool of HIV-1 in CSF. We utilized heteroduplex tracking assays to differentiate CSF HIV-1 variants that were shared with peripheral blood plasma from those that were compartmentalized in CSF and therefore presumably derived from local CNS tissues. We then tracked the relative decline of individual viral variants during the initial days of antiretroviral therapy. We found that HIV-1 variants compartmentalized in CSF declined rapidly during therapy, with maximum half-lives of approximately 1 to 3 days. These kinetics emulate the decline in HIV-1 produced from short-lived CD4+ T cells in the periphery, suggesting that a similarly short-lived, HIV-infected cell population exists within the CNS. We propose that short-lived CD4+ T cells trafficking between the CNS and the periphery play an important role in amplifying and maintaining HIV-1 populations in the CNS during the asymptomatic phase of infection.

AIDS Dementia Complex↗
PubMed2001

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Adult↗
PubMed2006

Cytomegalovirus encodes a positive regulator of antigen presentation.

Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.

Adoptive Transfer↗
PubMed1996

The role of a 21-kDa viral membrane protein in the assembly of vaccinia virus from the intermediate compartment.

We have recently provided morphological evidence that a key event in the assembly of vaccinia virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521-541). This tightly apposed cisternal domain incompletely surrounds the spherical immature virus that matures into the first of the two distinct infectious forms of vaccinia, the intracellular mature virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH2- and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies against Rab1. Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the IMV inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the IC and membranes of the viral factory.

Amino Acid Sequence↗
PubMed1997

Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.

The death of small intestinal epithelial cells has been characterized and quantitated after irradiation of mice rendered homozygously null for the p53 gene. In wild-type animals homozygous for p53 a rapid (4.5 h) elevation of p53 protein was observed in the proliferative compartment of the crypts after 8 Gy of irradiation. Cells underwent cell death by apoptosis in this region. We had reported previously a total repression of apoptosis in small intestinal crypt epithelia 4.5 h after the gamma-irradiation (8 Gy) of p53 homozygously null animals. Thus, while 400 apoptotic cells were observed in 200 half crypts taken from wild-type animals at 4.5 h, this fell to background levels (10-30) in the p53 null animals (Merritt et al., 1994) and did not increase by 12 h. However, we have now found a delayed initiation of a p53-independent apoptosis after 8 Gy of gamma-radiation: at 24 h, approximately 100 apoptotic cells were observed in 200 half crypts. This late wave of apoptosis was not observed after 1 Gy of gamma-radiation. The morphological appearance of this p53-independent apoptosis suggested that death may have arisen as the result of aberrant mitosis. Analysis of the regeneration of crypts 3 days after irradiation of mice with between 11 and 17 Gy showed that there was no significant increase (P=0.135) in the potential of clonogenic cells from the p53 null animals to repopulate the crypts. The data support the idea that a p53-independent apoptotic mechanism permits the engagement of apoptosis, probably by a mitotic catastrophe, after 8 Gy of gamma-irradiation in vivo and that a loss of p53 does not make these epithelial cells radioresistant in vivo to doses of 8 Gy and above. In contrast, irradiation with 1 Gy failed to induce a p53-independent apoptosis in vivo, suggesting that the p53 'sensor' of damage was more sensitive than that engaging the p53-independent mechanism of cell death.

Animals↗
PubMed1986

Regulation of hepatic growth.

The liver is a conditional renewal system, which in the adult organism undergoes minimal cell production and/or cell renewal. However, a reduction in liver cell mass, because of either actual cell loss or cell atrophy, evokes a rapid regenerative response tailored to replace the lost tissue. Synthesis of DNA begins as early as 15 h after a two-thirds hepatectomy, and the fact that all the remaining hepatocytes enter DNA synthesis within the next 48 h does indicate they are all potentially proliferative, and it is unlikely that a distinct stem cell compartment exists. The temporal sequelae of events can be best explained by the semisynchronous passage of cells from G0 into the proliferative cycle (see Fig. 2) where they undergo one or more rounds of cell division before decycling back into the proliferatively quiescent G0 state. The age of the animal and its nutritional and hormonal status are all important modifiers of the response, but none of them is critical to the regenerative process. Experiments involving the administration of sera or the transfer of blood between animals strongly favor the existence of humoral regulatory factors; the liver is apparently capable of producing both inhibitory and stimulatory molecules that act by negative and positive feedback mechanisms, respectively, to control tissue homeostasis, whereas other organs, notably the pancreas, are important sources of facilitatory molecules. A chemical mechanism of self inhibition is a very intellectually appealing hypothesis, but at present there is no consistent message as to the identity of the inhibitory molecule, although most studies suggest the target site for its action is the G1-S transition. Unless the whole field is one of multilateral analysis of an artifact, then endogenous growth inhibitors do exist, but the problem now is one of biochemical isolation and characterization. The field compares rather badly with the many success stories in recent years in which new hormones and peptides have been speedily isolated and purified. A reduction in liver size appears to be associated with a decrease in the concentration of an hepatic growth inhibitor and the production and/or unmasking of a stimulatory factor(s) that is also of hepatic origin. Once again, there is little information about the biochemical nature of the principle and much less on its mode of action. We all assume that such stimulators, and for that matter inhibitors as well, act on "restriction points" or "mitosis operons" and so on.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-Globulins↗
PubMed2005

Measles virus nucleoprotein induces cell-proliferation arrest and apoptosis through NTAIL-NR and NCORE-FcgammaRIIB1 interactions, respectively.

Measles virus (MV) nucleoprotein (N) is a cytosolic protein that is released into the extracellular compartment after apoptosis and/or secondary necrosis of MV-infected cells in vitro. Thus, MV-N becomes accessible to inhibitory cell-surface receptors: FcgammaRIIB and an uncharacterized nucleoprotein receptor (NR). MV-N is composed of two domains: NCORE (aa 1-400) and NTAIL (aa 401-525). To assess the contribution of MV-N domains and of these two receptors in suppression of cell proliferation, a human melanoma HT144 cell line expressing (HT144IIB1) or lacking FcgammaRIIB1 was used as a model. Specific and exclusive NCORE-FcgammaRIIB1 and NTAIL-NR interactions were shown. Moreover, NTAIL binding to human NR predominantly led to suppression of cell proliferation by arresting cells in the G0/G1 phases of the cell cycle, rather than to apoptosis. NCORE binding to HT144IIB1 cells primarily triggered caspase-3 activation, in contrast to HT144IIB1/IC- cells lacking the FcgammaRIIB1 intra-cytoplasmic tail, thus demonstrating the specific inhibitory effect of the NCORE-FcgammaRIIB1 interaction. MV-N- and NCORE-mediated apoptosis through FcgammaRIIB1 was inhibited by the pan-caspase inhibitor zVAD-FMK, indicating that apoptosis was dependent on caspase activation. By using NTAIL deletion proteins, it was also shown that the region of NTAIL responsible for binding to human NR and for cell growth arrest maps to one of the three conserved boxes (Box1, aa 401-420) found in N of Morbilliviruses. This work unveils novel mechanisms by which distinct domains of MV-N may display different immunosuppressive activities, thus contributing to our comprehension of the immunosuppressive state associated with MV infection. Finally, MV-N domains may be good tools to target tumour cell proliferation and/or apoptosis.

Animals↗
PubMed2005

The murine gammaherpesvirus 68 M2 gene is required for efficient reactivation from latently infected B cells.

Murine gammaherpesvirus 68 (gammaHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of gammaHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 x 10(5) PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD(-)) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD(-) B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD(-) B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.

Administration, Intranasal↗