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γ-Herpesviral immune evasion mechanisms are optimized to support the acute, lytic and the longterm, latent phase of infection. During acute infection, specific immune modulatory proteins limit, but also exploit, the antiviral activities of cell intrinsic innate immune responses as well as those of innate and adaptive immune cells. During latent infection, a restricted gene expression program limits immune targeting and cis-acting mechanisms to reduce the antigen presentation as well as antigenicity of latency-associated proteins. Here, we will review recent progress in our understanding of γ-herpesviral immune evasion strategies.

Human cytomegalovirus (HCMV) accelerates transplant vascular sclerosis (TVS), a consequence of angiogenesis (AG) and wound repair (WR). While HCMV can be localized to TVS lesions, the low number of infected cells suggests a global effect on target tissues. We used microarray analysis followed by real-time-polymerase chain reaction (RT-PCR) in an RCMV-accelerated TVS rat cardiac transplant model to determine whether CMV activates host WR and AG factors. Dysregulated cellular genes in allografts from RCMV-infected recipients were compared to those from uninfected recipients and native hearts. We demonstrated that RCMV upregulates the genes involved in WR and AG, which was highest during the critical time of TVS acceleration (21-28 days). Using a standard in vitro AG assay, virus and serum-free supernatants collected at 48 h postinfection significantly induced endothelial cell (EC) migration, branching and tubule formation compared to supernatants from mock-infected cells. Supernatants from ultraviolet (UV)-inactivated RCMV-infected cells failed to induce AG, indicating that virus replication is required. Upregulation of WR and AG genes occurs during the critical period of CMV-accelerated TVS. Targeting these genes may prevent this process and improve allograft survival.

CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules.

Virology research and antiviral drug discovery are poised to benefit from the post-genomic revolution for three main reasons. First, viruses need the host to replicate and are therefore vulnerable to inhibition of cellular pathways. Knowledge of complete genomic sequences of both virus and host now permits the study of this interplay on a global scale. Combining transcriptomics and proteomics with large-scale gene knockdown experiments will enable the identification of novel antiviral targets. Second, massive parallel assay systems, such as DNA microarrays, which define the post-genomic era, will facilitate viral diagnostics. Third, the combination of genetics with genomics will enable the analysis of viral mutants and strains on an unprecedented scale. The dramatic effects of viral infection on host cell transcriptional patterns have been well-documented and will be briefly highlighted. In addition, we discuss recent trends that apply functional genomics methods to the discovery of new targets and therapies for viral disease.

We have demonstrated that human brain capillary endothelial (HBCE) cells, unlike umbilical or aortic endothelial cells are permissively infected by HIV. HIV infection of HBCE cells is noncytolytic and is mediated by a CD4- and GalCer-independent mechanism, implying that HBCE cell tropic strains utilize a unique receptor. The V3 loop of gp120 appears to be important in this reaction. T-cell tropic but not brain-derived macrophage tropic HIV strains selectively infect brain endothelium suggesting that T-cell tropism is important for HIV entry through the blood-brain barrier (BBB). The ability of HIV to infect cells that compose the BBB implies that the virus may be directly involved in the BBB dysfunction observed in AIDS patients. HIV infection of HBCE cells may allow the flow of cytokines or toxic metabolites from the circulating blood into the brain parenchyma either by disrupting tight junctions or by altering the ability of the cells to regulate transport of substances across the BBB by transcytosis. HIV infection may also result in endothelial cell-induced astrocytosis by release of cytotoxic substances or modulation of abluminal surface antigens which contact astrocytic foot processes. Finally, HIV infection of the brain endothelium could facilitate virus entry to the CNS either by infection of HBCE cells or via entry of HIV-infected leucocytes. The establishment of our in vitro HIV-HBCE cell system will allow us to explore the potential mechanisms which mediate AIDS dementia.

IL-1-induced expression of granulopoietic factors, including G-CSF, by human bone marrow stromal cells is suppressed by HIV-1 infection, a phenomenon that may account for the regenerative granulopoietic failure often seen in HIV-1 infected patients. Seeking to identify the HIV-1 gene products that account for stromal cell dysfunction, we used replication incompetent HIV-1 mutants to demonstrate that deletion of vpr abrogates the stromal cell defect. Bone marrow stromal cell cultures were established in primaria dishes containing VEGF and were allowed to grow to near confluence. HIV-1 pseudotyped with VSV-G envelope protein was produced by transient co-transfection of 293T cells with a VSV-G expression vector and HIV-1 proviral plasmids containing: (1) a deletion in the envelope gene (env-) or (2) deletions of both env and vpr. Expression of G-CSF, GM-CSF, and IL-6 by stromal cells expressing the env deleted mutant was reduced by more than 50%, but this inhibitory effect was largely relieved by mutants with deletions of both env and vpr. To confirm the vpr effect, we carried out gain-of-function studies using bone marrow stromal cells transduced with the Moloney based expression vector pSLX-CMV- vpr or vector control pSLX-CMVX. IL-6 and G-CSF expression was reduced in stromal cells expressing vpr. We conclude that blunted responses of HIV-1 infected hematopoietic stromal cells depends, at least in part, upon the expression of HIV-1 vpr and that, in the case of G-CSF and IL-6, vpr is sufficient to account for this effect.

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10 6 . A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ∼ 1.3 × 10 6 . The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [ 14 C 6 ] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [ 14 C 6 ] glucose.

The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH 3 or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as γ-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed “dilution effect”. E. coli pro A − and pro B − auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes. In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of Δ 1 -pyrroline 5-carboxylate.

A study has been made of the experimental errors inherent in investigations of intermediary metabolism in a rat liver preparation, using C 14 -labeled substrates, chromatography, and radioautography as analytical tools. Assessed on the basis of absolute amount of C 14 incorporated into each metabolic product, the variation between experiments using samples of tissue from different animals was considerably greater than that using several samples of tissue from one animal. However, if the C 14 incorporated into each metabolite was expressed as a percentage of the total C 14 incorporated into all the metabolites, the variation between replicate samples of tissue from one animal was approximately the same as that using tissue from several animals. In both cases the experimental variation was such that, unless the effect of an alteration in the environment resulted in a change in the amount of C 14 in any particular compound of at least 80–85%, it could not be regarded as significant. This applied only to compounds containing more than 2% of the total incorporated C 14 ; substances containing less C 14 showed an even greater variation. The main sources of variation were probably slight uncontrolled differences in the incubation conditions, together with inaccuracies in the determinations of radioactivity in those compounds containing very small amounts of the radioisotope.

1. 1. Biochemical compartmentation, in the sense of intermediates in a pathway not being freely miscible with other cellular pools of the same compounds, was investigated in histidine biosynthesis in Salmonella typhimurium and Saccharomyces cerevisiae . 2. 2. The experimental approach involved measurement of the degree of dilution by unlabelled histidinol, added to the medium, of 14 C incorporated endogenously from labelled glucose into the histidine of the cell proteins. 3. 3. Compartmentation, on this basis, was absent in Salm. typhimurium but present in Sacc. cerevisiae . 4. 4. In Sacc. cerevisiae , the absence of competition between internally formed and externally added histodinol was not due to a failure of this substance to penetrate the cell membrane.

In view of the wide application of radioactive carbon dioxide in tracer experiments together with a reported anomalous effect of the isotope content on the tracer behavior, we have investigated the effect of varying specific activity of the carbon dioxide on both the rate and pattern of photosynthetic carbon fixation. We have found no effect of the carbon-14 other than the ordinary mass effect in both algae and higher plants.

Using radioactive carbon dioxide, an attempt has been made to distinguish the various pools of intermediary metabolism which may be physically or chemically separate within the cell. Some correlation between the structural elements of the cells and these pools appears possible.