Modulate Host Gene Expression Research Articles

Bacterium-Generated Nitric Oxide Hijacks Host Tumor Necrosis Factor Alpha Signaling and Modulates the Host Cell Cycle In Vitro

In mammalian cells, nitric oxide (NO·) is an important signal molecule with concentration-dependent and often controversial functions of promoting cell survival and inducing cell death. An inducible nitric oxide synthase (iNOS) in various mammalian cells produces higher levels of NO· from l-arginine upon infections to eliminate pathogens. In this study, we reveal novel pathogenic roles of NO· generated by bacteria in bacterium-host cell cocultures using Moraxella catarrhalis, a respiratory tract disease-causing bacterium, as a biological producer of NO·. We recently demonstrated that M. catarrhalis cells that express the nitrite reductase (AniA protein) can produce NO· by reducing nitrite. Our study suggests that, in the presence of pathophysiological levels of nitrite, this opportunistic pathogen hijacks host cell signaling and modulates host gene expression through its ability to produce NO· from nitrite. Bacterium-generated NO· significantly increases the secretion of tumor necrosis factor alpha (TNF-α) and modulates the expression of apoptotic proteins, therefore triggering host cell programmed death partially through TNF-α signaling. Furthermore, our study reveals that bacterium-generated NO· stalls host cell division and directly results in the death of dividing cells by reducing the levels of an essential regulator of cell division. This study provides unique insight into why NO· may exert more severe cytotoxic effects on fast growing cells, providing an important molecular basis for NO·-mediated pathogenesis in infections and possible therapeutic applications of NO·-releasing molecules in tumorigenesis. This study strongly suggests that bacterium-generated NO· can play important pathogenic roles during infections.

Small RNAs containing the pathogenic determinant of a chloroplast‐replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

A Novel PA-X Protein Translated from Influenza A Virus Segment 3

The pathogenicity of influenza A viruses is a multigenic trait, which is orchestrated by the global networks between eight viral genomic constituents and their cellular interacting partners. A recent report provided information on the finding of a new PA ribosomal frameshifting product, the PA-X protein, in the influenza A virus segment 3, and an endonuclease property was suggested for a possible role of the PA-X protein. In cultured cells, viral growth was not affected by the PA-X protein expression. However, the reduced pathogenicity of mice appeared to be closely associated with the PA-X protein expression. It was also revealed that the PA-X protein was able to modulate host gene expression. Considered together, the PA-X protein can be a cellular signaling modulator and subsequently control viral pathogenicity. By reviewing recent publications, we present new insights in the contribution of the PA-X protein to the cellular signaling network and the resultant viral pathogenicity.

Activation of LTRs from Different Human Endogenous Retrovirus (HERV) Families by the HTLV-1 Tax Protein and T-Cell Activators

Human endogenous retroviruses (HERVs) represent approximately 8% of our genome. HERVs influence cellular gene expression and contribute to normal physiological processes such as cellular differentiation and morphogenesis. HERVs have also been associated with certain pathological conditions, including cancer and neurodegenerative diseases. As HTLV-1 causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has been shown to modulate host gene expression mainly through the expression of the powerful Tax transactivator, herein we were interested in looking at the potential modulation capacity of HTLV-1 Tax on HERV expression. In order to evaluate the promoter activity of different HERV LTRs, pHERV-LTR-luc constructs were co-transfected in Jurkat T-cells with a Tax expression vector. Tax expression potently increased the LTR activity of HERV-W8 and HERV-H (MC16). In parallel, Jurkat cells were also stimulated with different T-cell-activating agents and HERV LTRs were observed to respond to different combination of Forskolin, bpV[pic] a protein tyrosine phosphatase inhibitor, and PMA. Transfection of expression vectors for different Tax mutants in Jurkat cells showed that several transcription factors including CREB appeared to be important for HERV-W8 LTR activation. Deletion mutants were derived from the HERV-W8 LTR and the region from −137 to −123 was found to be important for LTR response following Tax expression in Jurkat cells, while a different region was shown to be required in cells treated with activators. Our results thus demonstrated that HTLV-1 Tax activates several HERV LTRs. This raises the possibility that upregulated HERV expression could be involved in diseases associated with HTLV-1 infection.

Microbiota Modulate Host Gene Expression via MicroRNAs

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3′-untranslated regions (3′-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3′-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.

Phosphorylation and Polyubiquitination of Transforming Growth Factor β-Activated Kinase 1 Are Necessary for Activation of NF-κB by the Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor

Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) protein has been shown to induce several signaling pathways leading to the modulation of host gene expression. The hijacking of these pathways facilitates the viral life cycle and leads to tumorigenesis. In the present work, we show that transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is an important player in NF-κB activation induced by vGPCR. We observed that the expression of an inactive TAK1 kinase mutant (TAK1M) reduces vGPCR-induced NF-κB nuclear translocation and transcriptional activity. Consequently, the expression of several NF-κB target genes normally induced by vGPCR was blocked by TAK1M expression, including interleukin 8 (IL-8), Gro1, IκBα, COX-2, cIAP2, and Bcl2 genes. Similar results were obtained after downregulation of TAK1 by small interfering RNA (siRNA) technology. The expression of vGPCR recruited TAK1 to the plasma membrane, and vGPCR interacts with TAK1. vGPCR expression also induced TAK1 phosphorylation and lysine 63-linked polyubiquitination, the two markers of the kinase's activation. Finally, inhibition of TAK1 by celastrol inhibited vGPCR-induced NF-κB activation, indicating this natural compound could be used as a potential therapeutic drug against KSHV malignancies involving vGPCR.

Role of gut microbiota in the control of energy and carbohydrate metabolism

To describe the recent developments and insights gained in the role played by the colonic microbiota in energy and carbohydrate metabolism related to obesity in humans. Previous findings that the ratio of Firmicutes and Bacteriodetes is important in energy harvesting and obesity have not been confirmed in recent studies. In fact, sometimes, the opposite results were obtained. Nevertheless, it is clear that the microbiota plays a role in energy extraction from nondigested carbohydrates in the form of production of short-chain fatty acids. Also, the microbiota plays a role in host metabolism by influencing and modulating host gene expression in various tissues. Despite numerous recent studies trying to link the composition of the microbiota to obesity, the picture is far from clear, and it remains to be seen whether changes in microbiota composition are the cause or the consequence of obesity. Molecular studies reveal the enzyme machineries used by individual members of the microbiota to break down and ferment polysaccharides. Also, the mechanisms of host-microbe mutualism are becoming unraveled. Using stable-isotope-labeled substrates, the exact microorganisms involved in fermentation of the substrates and the exact metabolites that are produced from the substrate can be deciphered.

Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1

Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C-X-C and C-C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.

Impaired Replication of Hepatitis C Virus Containing Mutations in a Conserved NS5B Retinoblastoma Protein-Binding Motif

Hepatitis C virus (HCV) downregulates the retinoblastoma tumor suppressor protein (Rb), a central cell cycle regulator which is also targeted by oncoproteins expressed by DNA tumor viruses. HCV genome replication is also enhanced in proliferating cells. Thus, it is possible that HCV interactions with host cell cycle regulators, such as Rb, have evolved to modify the intracellular environment to promote viral replication. To test this hypothesis and to determine the impact of viral regulation of Rb on HCV replication, we constructed infectious viral genomes containing mutations in the Rb-binding motif of NS5B which ablate the ability of HCV to regulate Rb. These genomes underwent replication in transfected cells but produced variably reduced virus yields. One mutant, L314A, was severely compromised for replication and rapidly mutated to L314V, thereby restoring both Rb regulation and replication competence. Another mutant, C316A, also failed to downregulate Rb abundance and produced virus yields that were about one-third that of virus with the wild-type (wt) NS5B sequence. Despite this loss of replication competence, purified NS5B-C316A protein was two- to threefold more active than wt NS5B in cell-free polymerase and replicase assays. Although small interfering RNA knockdown of Rb did not rescue the replication fitness of these mutants, we conclude that the defect in replication fitness is not due to defective polymerase or replicase function and is more likely to result from the inability of the mutated NS5B to optimally regulate Rb abundance and thereby modulate host gene expression.

Microarray study reveals that HIV-1 induces rapid type-I interferon-dependent p53 mRNA up-regulation in human primary CD4+T cells

BackgroundInfection with HIV-1 has been shown to alter expression of a large array of host cell genes. However, previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions, we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix.ResultsWe report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair, cellular cycle, RNA metabolism and apoptosis. Notably, expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level, which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-α and IFN-β).ConclusionThese observations have important implications for our understanding of HIV-1 pathogenesis, particularly in respect to the virus-induced depletion of CD4+ T cells.

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

R5 and X4 HIV Viruses Differentially Modulate Host Gene Expression in Resting CD4+T Cells

During HIV-1 infection, distinct biological phenotypes are observed between R5 and X4 HIV-1 strains with respect to pathogenicity and tropism. In this study, temporal changes of the expression levels of the complete human transcriptome, representing 47,000 well-characterized human transcripts, were monitored in the first 24 h during HIV-1 R5 and X4 exposition in resting primary CD4(+) T cells. We provide evidence that R5 viruses modulate, to a greater extent than X4 viruses, the level of mRNA of the resting CD4(+) T cells. Indeed, modulation of the TCR signaling and the actin organization involving the WAVE/ABI complex and the ARP2/3 complex appeared to be associated with R5 exposition. The data suggest that the ability of R5 viruses to modulate TCR-mediated actin polymerization and signaling creates a favorable environment for CD4(+) T cell activation after TCR stimulation and may partly explain why R5 is the primary strain observed early in the natural infection process.

Modulation of Gene Expression in Human Macrophages Treated with the Anti- Leishmania Pentavalent Antimonial Drug Sodium Stibogluconate

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan parasite that resides and multiplies exclusively in the phagolysosomes of macrophages. Leishmania control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam) or meglumine antimoniate (Glucantime). The mode of action of SbV is still not known precisely. To explore the effect of SbV on macrophage gene expression, a microarray analysis was performed using Affymetrix focus arrays to compare gene expression profiles in noninfected and L. donovani-infected THP-1 monocytic cells treated or not treated with sodium stibogluconate. Under our experimental conditions, SbV changed the expression of a few host genes, and this was independent of whether cells were infected or not infected with Leishmania. Leishmania infection had a greater effect on the modulation of host gene expression. Statistical analyses have indicated that the expression of eight genes was modified by at least twofold upon SbV treatment, with six genes upregulated and two genes downregulated. One gene whose expression was affected by SbV was the heme oxygenase gene HMOX-1, and this change was observed both in the monocytic cell line THP-1 and in primary human monocyte-derived macrophages. Another pathway that was affected was the glutathione biosynthesis pathway, where the expression of the glutamate-cysteine ligase modifier subunit was increased upon SbV treatment. Our analysis has suggested that, under our experimental conditions, the expression of a few genes is altered upon SbV treatment, and some of these encoded proteins may be implicated in the yet-to-be-defined mode of action of SbV.

Modulation of Host Gene Expression by the K15 Protein of Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) encoding proteins capable of initiating signal transduction pathways. Among them is the K15 ORF, which consists of eight exons encoding a protein with 12 predicted transmembrane domains and a cytoplasmic C terminus. When transiently expressed, the 8-exon K15 transcript gives rise to a protein with an apparent molecular mass of 45 kDa. K15 interacts with cellular proteins, TRAF (tumor necrosis factor receptor-associated factor) and Src kinases, and activates AP-1, NF-kappaB, and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase and extracellular signal-regulated kinase. This signaling activity of K15 is related to phosphorylation of Y(481) of the K15 SH2-B motif Y(481)EEV. In this study we demonstrate the expression of an endogenous 45-kDa K15 protein in KSHV BAC36-infected epithelial cells. This endogenous K15 protein shows the same intracellular localization as transiently expressed K15, and expression kinetic studies suggest it to be a lytic gene. We have further determined the downstream target genes of K15 signaling using DNA oligonucleotide microarrays. We demonstrate that K15 is capable of inducing expression of multiple cytokines and chemokines, including interleukin-8 (IL-8), IL-6, CCL20, CCL2, CXCL3, and IL-1alpha/beta, as well as expression of Dscr1 and Cox-2. In epithelial cells, K15-induced upregulation of most genes was dependent on phosphorylation of Y(481), whereas in endothelial cells mutation of Y(481) did not result in a complete loss of Dscr1 and Cox-2 expression and NFAT-activity. Our study establishes K15 as one of the KSHV lytic genes that are inducing expression of multiple cytokines, which have been shown to play an important role in KSHV-associated pathogenesis.

Hepatitis B virus replication causes oxidative stress in HepAD38 liver cells

We used human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promotor, to investigate changes induced in the host cell by HBV replication that could contribute to malignant transformation. Parameters of oxidative stress (malondialdehyde, glutathione) and cell proliferation were determined at different times after induction (0-96 h). In HBV-producing cells, the redox status peaked at 72 h. cDNA micro array analysis at 72 h post induction revealed 3 groups of genes that were up-regulated by HBV: (i) heat shock proteins, (ii) oxidative and metabolic stress and (iii) growth and apoptosis related genes. Continuous HBV production did not accelerate karyotypic changes in cells cultured for 4 months (18 passages). HBV replication modulates host gene expression and induces oxidative stress. In this HepAD38 model early events (0-4 days) in the host cell after induction of HBV replication can be studied under strictly defined conditions.

Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression

BackgroundCnidarian – dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian – algal symbiosis.ResultsWe detected statistically significant differences in host gene expression profiles between sea anemones (Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian – dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress.ConclusionOur data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian – dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

Modulation of Host Gene Expression and Innate Immunity by Viruses

Viruses characteristically infect host cells directly and have tremendously rapid replicative capacity. For these reasons, an organism’s earliest defenses against viruses are essential for host survival. These responses are referred to as innate and represent the entire repertoire of immunity in phylogenetically less-complex organisms. Innate responses are capable of participating in defense quickly and without the need for rearranging germline DNA to create specificity. Modulation of Host Gene Expression and Innate Immunity by Viruses presents a state-of-the art dissection of these defense mechanisms as well as strategies that viruses have developed to evade them. Furthermore, influences that viruses have upon host gene expression favoring either the host or the virus are also recounted in the context of innate immunity.

Upregulation of functionally active vascular endothelial growth factor by human cytomegalovirus

Human cytomegalovirus (HCMV) infection is known to modulate host gene expression and has been linked to the pathogenesis of vasculopathies; however, relevant pathomechanisms are still unclear. It was shown that HCMV infection leads to upregulation of vascular endothelial growth factor (VEGF) expression in human foreskin fibroblasts and coronary artery smooth muscle cells (SMC). Activation of VEGF transcription by HCMV infection was confirmed by transient-expression experiments, which revealed that a short promoter fragment, pLuc135 (-85 to +50), is sufficient for activation. Site-directed mutagenesis of Sp1-recognition sites within this fragment abolished the upregulation of transcription. Functional VEGF protein is released into the culture supernatant of infected SMC. Incubation of endothelial cells with supernatants from HCMV-infected SMC cultures induced upregulation of VEGF receptor-2 expression on endothelial cells, as well as a significant upregulation of DNA synthesis, implicating cell proliferation. The mean incline of DNA synthesis at 48 and 72 h post-infection was 148 and 197 %, respectively. Addition of neutralizing antibodies against VEGF completely abolished this effect. Supernatants from SMC cultures incubated with UV-inactivated virus induced a comparable effect. This virus-induced paracrine effect may represent a molecular mechanism for HCMV-induced pathogenesis, such as inflammatory vasculopathies, by inducing a proatherogenic phenotype in SMC.

A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein.

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.

Host Factors in Positive-Strand RNA Virus Genome Replication

All viruses are gene poor relative to their hosts: even the largest viral genomes only encode hundreds of genes, while those of host cells typically encode tens of thousands of genes. Thus, most steps in virus infection involve interactions between relatively few different types of viral components and much more complex pools of host factors. This sea of host factors represents both the essential milieu to which viruses must adapt for survival and a tremendous, manipulatable resource for gene-poor viruses. Accordingly, host factors play important roles in most steps of viral infection, and identifying such host factors and their contributions has long been recognized as an important frontier. The continuing emergence of close integration between viral and host functions in infection suggests moving beyond separate views of virus and host to a more holistic view of the virus-infected cell as a unified entity that constitutes the functional unit of infection. One area in which the importance of host factors is increasingly emerging is the replication of positive-strand RNA viruses. Positive-strand RNA viruses encompass over one-third of all virus genera and include numerous pathogens, such as the severe acute respiratory syndrome coronavirus SARS, hepatitis C virus (HCV), and many of the viruses on the U.S. Health and Human Services Department Select List of potential bioterrorism agents. Host factors participate in most, if not all, steps of positive-strand RNA virus infection, including entry, viral gene expression, virion assembly, and release. Moreover, host factors are targeted by positive-strand RNA viruses to modulate host gene expression and defenses. This review focuses on host factors involved in positivestrand RNA virus genome replication. The evidence for such host factor involvement has come from varied genetic and biochemical approaches (15). These include, among others, studies based on the varying permissiveness of some cell types and extracts for RNA replication (2, 3, 4, 19); identification of many host proteins that interact with viral genomic RNAs or replication proteins and, in some cases, have been functionally linked to replication (5, 18, 27, 35, 37, 40); and mutational screens in genetic model systems, such as Arabidopsis thaliana (17, 39) and the yeast Saccharomyces cerevisiae (8, 11, 16, 23, 38). Recent data show that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral RNA replication templates, activating the complex for RNA synthesis, and other steps. Each of these virushost interactions may contribute to the host specificity, tissue specificity, or pathology of infections. Each such virus-host interaction also represents a potential target for virus control or for optimization to improve beneficial uses of viruses and their components. Positive-strand RNA viruses can be divided into a number of superfamilies defined by distinguishable RNA replication genes and features (41). Nevertheless, the RNA replication mechanisms of these viruses share sufficient similarities to make it reasonable to discuss their replication as a class and to attempt to draw general lessons by comparing examples from different superfamilies. As discussed further below, a few of the common features shared by positive-strand RNA viruses are the need to coordinate use of the infecting viral genomic RNA as a template for translation and replication, assembly of replication complexes on intracellular membranes, and production of 10- to 100-fold excesses of positive- over negativestrand RNA. A simplified general scheme for RNA replication by positive-strand RNA viruses is shown in Fig. 1. Below we discuss the involvement of host factors in the various stages of this RNA replication process, using examples from a number of viruses. We regret that space limitations do not allow us to cite all of the work being done in this exciting area.