Institute of Molecular Biology

Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization, which contributed to a decline in their pursuit by the pharmaceutical industry from the 1990s onwards. In recent years, several technological and scientific developments - including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances - are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized, particularly for tackling antimicrobial resistance. Here, we summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities.

BACKGROUND: The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown. METHODS: We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro. RESULTS: Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P<0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib. CONCLUSIONS: Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.

A package of programs has been developed for efficient restrained least-squares refinement of macromolecular crystal structures. The package has been designed to be as flexible and general purpose as possible. The process of refinement is divided into basic units and an independent computer program handles each task. Each functional unit communicates with other programs in the package by way of files of well defined format. To modify or replace any program, the user need only understand the function of that particular element. Stereochemical restraints are defined in a general way that can be applied to proteins, nucleic acids, prosthetic groups, solvent atoms and so on. Guide values for bond lengths and bond angles are specified in a straightforward direct manner. Designated groups of atoms can be held constant or constrained to behave as a rigid body during refinement. In order to make the package as efficient as possible, the fast Fourier transform algorithm is used for all the crystallographic transformations. To highlight potential errors in the refined structure the user can list those atoms that have the worst bond lengths and angles, or have the largest positional, temperature-factor or occupancy gradients. It is also possible to check that protein and solvent atoms do not sterically clash with symmetry-related neighbors. Applications of the program package to a bacteriochlorophyll-containing protein, thermolysin-inhibitor complexes and mutants of bacteriophage T4 lysozyme are described.

BACKGROUND: Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS: Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS: Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS: Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

Alzheimer’s disease (AD) is the most common cause of progressive dementia in the elderly. It is characterized by a progressive and irreversible loss of cognitive abilities and formation of senile plaques, composed mainly of amyloid β (Aβ), and neurofibrillary tangles (NFTs), composed of tau protein, in the hippocampus and cortex of afflicted humans. In brains of AD patients the metabolism of Aβ is dysregulated, which leads to the accumulation and aggregation of Aβ. Metabolism of Aβ and tau proteins is crucially influenced by autophagy. Autophagy is a lysosome-dependent, homeostatic process, in which organelles and proteins are degraded and recycled into energy. Thus, dysfunction of autophagy is suggested to lead to the accretion of noxious proteins in the AD brain. In the present review, we describe the process of autophagy and its importance in AD. Additionally, we discuss mechanisms and genes linking autophagy and AD, i.e., the mTOR pathway, neuroinflammation, endocannabinoid system, ATG7, BCL2, BECN1, CDK5, CLU, CTSD, FOXO1, GFAP, ITPR1, MAPT, PSEN1, SNCA, UBQLN1, and UCHL1. We also present pharmacological agents acting via modulation of autophagy that may show promise in AD therapy. This review updates our knowledge on autophagy mechanisms proposing novel therapeutic targets for the treatment of AD.

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.

Regulation of the GAL structural genes in the yeast Saccharomyces cerevisiae is implemented by the products of GAL-specific (GAL4, GAL80, GAL3) and general (GAL11, SWI1, 2, 3, SNF5, 6, numerous glucose repression) genes. Recent work has 1) yielded significant new insights on the DNA binding and transcription activation/Gal80 protein binding functions of the Gal4 activator protein, 2) described the characterization of purified Gal4 protein-Gal80 protein complexes, 3) deconvoluted the multiple and complex glucose repression pathways acting on GAL genes, 4) suggested a new mechanism for the Gal3 protein-mediated induction of GAL structural gene expression, 5) introduced Gal1 protein, a structural gene product, into the regulation scheme, and 6) extended our already substantial understanding of GAL regulatory gene control. The mechanisms which control structural and regulatory gene expression in the GAL family are compared and GAL structural/regulatory gene chromatin structure is discussed.

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position -87 and CRE-2 at -70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain (NICD) that functions directly in signal transduction. The Notch receptor is locked down in a protease-resistant state by a negative regulatory region (NRR) that protects an ADAM (a disintegrin and metalloprotease) cleavage site. Engagement with ligand-bearing cells induces global conformational movements in Notch that unfold the NRR structure to expose the ADAM cleavage site and initiate proteolytic activation. Although both ADAM10 and ADAM17 have been reported to cleave Notch to facilitate NICD release by gamma-secretase, the relevant ADAM has remained controversial. Our study provides new insight into this conflict, as we find that although Notch1 (N1) is a substrate for both ADAM10 and ADAM17, the particular ADAM required for receptor activation is context dependent. Specifically, ADAM10 was absolutely required for N1 signaling induced by ligands, while signaling independent of ligands required ADAM17. In contrast to the strict and differential use of ADAM10 and ADAM17 in normal and dysregulated signaling, respectively, both proteases participated in signaling intrinsic to N1 mutations associated with leukemia. We propose that in addition to exposing the ADAM cleavage site, activating N1 conformational changes facilitate selective cleavage by specific proteases.

A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.

The genetic contribution to the variation in human lifespan is ∼ 25%. Despite the large number of identified disease-susceptibility loci, it is not known which loci influence population mortality. We performed a genome-wide association meta-analysis of 7729 long-lived individuals of European descent (≥ 85 years) and 16 121 younger controls (<65 years) followed by replication in an additional set of 13 060 long-lived individuals and 61 156 controls. In addition, we performed a subset analysis in cases aged ≥ 90 years. We observed genome-wide significant association with longevity, as reflected by survival to ages beyond 90 years, at a novel locus, rs2149954, on chromosome 5q33.3 (OR = 1.10, P = 1.74 × 10(-8)). We also confirmed association of rs4420638 on chromosome 19q13.32 (OR = 0.72, P = 3.40 × 10(-36)), representing the TOMM40/APOE/APOC1 locus. In a prospective meta-analysis (n = 34 103), the minor allele of rs2149954 (T) on chromosome 5q33.3 associates with increased survival (HR = 0.95, P = 0.003). This allele has previously been reported to associate with low blood pressure in middle age. Interestingly, the minor allele (T) associates with decreased cardiovascular mortality risk, independent of blood pressure. We report on the first GWAS-identified longevity locus on chromosome 5q33.3 influencing survival in the general European population. The minor allele of this locus associates with low blood pressure in middle age, although the contribution of this allele to survival may be less dependent on blood pressure. Hence, the pleiotropic mechanisms by which this intragenic variation contributes to lifespan regulation have to be elucidated.

Recent studies have demonstrated that discriminatory salivary biomarkers can be readily detected upon the development of systemic diseases such as pancreatic cancer, breast cancer, lung cancer, and ovarian cancer. However, the utility of salivary biomarkers for the detection of systemic diseases has been undermined due to the absence of the biological and mechanistic rationale as to why distal diseases from the oral cavity would lead to the development of discriminatory biomarkers in saliva. Here, we examine the hypothesis that pancreatic tumor-derived exosomes are mechanistically involved in the development of pancreatic cancer-discriminatory salivary transcriptomic biomarkers. We first developed a pancreatic cancer mouse model that yielded discriminatory salivary biomarkers by implanting the mouse pancreatic cancer cell line Panc02 into the pancreas of the syngeneic host C57BL/6. The role of pancreatic cancer-derived exosomes in the development of discriminatory salivary biomarkers was then tested by engineering a Panc02 cell line that is suppressed for exosome biogenesis, implanting into the C56BL/6 mouse, and examining whether the discriminatory salivary biomarker profile was ablated or disrupted. Suppression of exosome biogenesis results in the ablation of discriminatory salivary biomarker development. This study supports that tumor-derived exosomes provide a mechanism in the development of discriminatory biomarkers in saliva and distal systemic diseases.

A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.

The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high-density lipoproteins (HDLs) and HDL-associated alpha-tocopherol (alpha TocH) across an in vitro model of the blood-brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL-associated alpha TocH was taken up in 10-fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR-BI). SR-BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl-beta-cyclodextrin (CDX) resulted in (mis)sorting of SR-BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR-BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL-associated [(14)C]alpha TocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin-1, the major caveolae-associated protein. During mass transfer studies using alpha TocH-enriched HDL, approximately 50% of cellular alpha TocH was recovered with the bulk of cellular caveolin-1 and SR-BI. Efflux experiments revealed that a substantial amount of cell-associated [(14)C]alpha TocH could be mobilized into the culture medium. In addition, apical-to-basolateral transport of HDL holoparticles and HDL-associated alpha TocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A-I and alpha TocH originates from plasma HDL transcytosed across the BBB and that caveolae-located SR-BI facilitates selective uptake of HDL-associated alpha TocH at the BBB.

Nasopharyngeal carcinoma, Kaposi's sarcoma, and B-cell lymphomas are human malignancies associated with gammaherpesvirus infections. Members of this virus family are characterized by their ability to establish latent infections in lymphocytes. The latent viral genome expresses very few gene products. The infected cells are therefore poorly recognized by the host immune system, allowing the virus to persist for long periods of time. We sought to identify the cell-specific factors that allow these viruses to redirect their life cycle from productive replication to latency. We find that the cellular transcription factor NF-kappaB can regulate this process. Epithelial cells and fibroblasts support active (lytic) gammaherpesvirus replication and have low NF-kappaB activity. However, overexpression of NF-kappaB in these cells inhibits the replication of the gammaherpesvirus murine herpesvirus 68 (MHV68). In addition, overexpression of NF-kappaB inhibits the activation of lytic promoters from MHV68 and human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). In lymphocytes latently infected with KSHV or EBV, the level of NF-kappaB activity is high, and treatment of these cells with an NF-kappaB inhibitor leads to lytic protein synthesis consistent with virus reactivation. These results suggest that high levels of NF-kappaB can inhibit gammaherpesvirus lytic replication and may therefore contribute to the establishment and maintenance of viral latency in lymphocytes. They also suggest that NF-kappaB may be a novel target for the disruption of virus latency and therefore the treatment of gammaherpesvirus-related malignancies.

During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-alpha 1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.