Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the Major Capsid Protein

A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and -sensitive strains. In a plaque reduction assay under conditions in which the 50% effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25 μM, the 50% effective concentration of 35B2 was 0.75 μM. The selective index of the compound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open reading frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corresponding to the “floor” domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electron microscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are required to clarify the precise antiviral mechanism.     

Papillomavirus Assembly Requires Trimerization of the Major Capsid Protein by Disulfides between Two Highly Conserved Cysteines

We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids.     

Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.     

Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.     

Identification and Fine Mapping of Nuclear and Nucleolar Localization Signals within the Human Ribosomal Protein S17

Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.     

Synthesis and Assembly of Simian Virus 40 II. Synthesis of the Major Capsid Protein and Its Incorporation into Viral Particles

African green monkey kidney cells infected by simian virus 40 were analyzed for the presence of the major capsid protein (capsid protein I) by immunological and radiolabeling techniques. Antisera with different specificities were prepared by immunization with intact or denatured viral particles. Antisera prepared against intact virus reacted by complement fixation with viral particles and with an 8S subunit containing the capsid protein I. Antisera prepared against denatured viral particles reacted with unassembled capsid protein(s) as well as with viral particles. These antisera were used to detect 8S viral subunits or unassembled viral capsid protein in soluble extracts of infected cells after centrifugation at 100,000 x g to remove viral particles. The soluble antigen pool was found to be small during infection with wild-type virus or a temperature-sensitive mutant deficient in the synthesis of viral particles. Pulse-chase experiments, performed at a high multiplicity of infection, also indicated a small pool of nonparticle capsid protein I. Radioactive lysine was incorporated into capsid protein I of virus particles during a 2-hr pulse. A subsequent chase with excess unlabeled lysine resulted in only a slight increase in the radio-activity found in capsid protein I of viral particles. Furthermore, in the same experiments, capsid protein I was incorporated preferentially into empty shells during the pulse with a shift in radioactivity to intact virions during the chase period, indicating a possible precursor relationship between the two types of virus particles.     

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site.     

Nuclear Import and Export Signals Control the Subcellular Localization of Nurr1 Protein in Response to Oxidative Stress

Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.     

Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT

The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14(bIL170)]), orf41 (P008 [orf41(P008)]), and orf6 (p2 [orf6(p2)] and P008 [orf6(P008)]). The genes e14(bIL170) and orf41(P008) are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.     

Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.Our understanding of herpesvirus morphogenesis is mainly derived from studies of Alphaherpesvirinae, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV). A faster replication cycle and a more productive infection in tissue culture aided genetic analysis of alphaherpesvirus morphogenesis. In addition, deletion mutants of key morphogenesis genes in alphaherpesviruses often maintain basic replication capacity, whereas the mutations of their homologues in Betaherpesvirinae or Gammaherpesvirinae mostly result in a lethal phenotype (for the UL31 and the UL34 family, see references 3, 6, 9-11, 16, 20, 21, and 42). These genes became amenable to comprehensive genetic analysis in betaherpesviruses only after their genomes were cloned as infectious bacterial artificial chromosomes (BACs), which obviated the need to generate replication-competent intermediates or complementing cell lines (3, 21, 23). BAC-based mutagenesis allowed viability screens mapping essential genes (8, 43) or even functional sites of essential genes in cytomegaloviruses (3, 21). However, these approaches cannot easily be applied to reveal the null phenotypes in the context of virus replication, as mutant viruses are not easily reconstituted. In addition, deletion of an essential viral gene can reveal the null phenotype of only the first of perhaps several essential functions during virus morphogenesis. This problem can be addressed to some extent by using dominant negative (DN) mutations (36). DN mutants are loss-of-function mutants that induce a null phenotype in the presence of the wild-type (wt) allele (14). Analysis of phenotypes induced by DN mutants proved to be extremely useful in genetics and cell biology, signaling, and biochemistry. Such inhibitory mutants of cellular proteins are often designed based on knowledge on the structural or functional role of a well-characterized protein domain. Unfortunately, we lack the structural information that would allow knowledge-based design of viral DN mutants for the majority of herpesvirus gene products. Thus, we established a random screen consisting of three steps to identify mutants of viral genes with DN potential (36): (i) a library of mutants is generated by random insertion of 5 amino acids (aa) or a stop codon into the open reading frame (ORF) of interest using transposon mutagenesis, (ii) nonfunctional mutants are identified by cis complementation of the respective deletion mutant mouse cytomegalovirus (MCMV) BAC, and (iii) nonfunctional mutants are tested for their inhibitory potential upon reconstitution of the wt BAC cloned genomes. In the last screen, mutants that have a specific inhibitory effect on the activity of the wt allele are selected. The specific phenotype obtained upon induction of the inhibitory mutants in the context of virus replication is then verified and further characterized using a tetracycline (Tet) regulon-based viral conditional expression system (36, 37).One intriguing aspect of herpesvirus morphogenesis is the transition of capsids from the nuclear to the cytoplasmic phase of virus morphogenesis. Two conserved nonstructural proteins, the homologues of the membrane protein pUL34 and its nuclear partner protein pUL31, form a nuclear egress complex (NEC) (18, 27, 42), which is required for primary envelopment and export of nuclear capsids to the cytoplasm (reviewed in references 24 and 25). Recent studies have revealed that the homologues of alphaherpesvirus pUL34 and pUL31, the M50 and the M53 gene products of the betaherpesvirus MCMV (pM50 and pM53, respectively) and the BFRF1 and the BFLF2 gene products of the gammaherpesvirus Epstein-Barr virus (EBV), apparently share the major functions of these two proteins. The lack of one or both proteins of the NEC generally results in the retention of viral capsids in the nucleus. This is lethal for beta- and gammaherpesvirus production (3, 9-11, 16, 18, 21, 27, 35, 42).The details of the mechanisms by which the NEC proteins mediate capsid export through the nuclear envelope are poorly understood. We (3, 21, 36, 38) and others (1, 19, 34) have started to dissect details of the NEC function using a genetic approach based on subtle mutagenesis of the respective genes. Analysis of the MCMV M50 gene by comprehensive mutagenesis localized two different functional sites. They were the M53 binding site within the N-terminal domain of M50, as well as the transmembrane region at its C terminus (3). Liang and Baines located the respective binding site in HSV-1 UL34 at aa 137 to 181 (19). Our approach, based on screens for DN mutants, identified a proline-rich sequence (aa 179 to 207) in the M50 gene product as an additional essential region (36). A recombinant virus expressing an M50 mutant lacking this site was defective in capsid egress from the nucleus despite the presence of the wt M50 protein. Consequently, the production of infectious particles after infection was reduced by more than 2 orders of magnitude. The UL34 homologues of alpha- and gammaherpesviruses lack a similar polyproline motif, but the result was confirmed by mutating the human cytomegalovirus (HCMV) homologue UL50 at the corresponding region, which is conserved within betaherpesviruses (36). The M50 mutants lacking the proline-rich motif still bind and colocalize to their respective NEC partner, pM53. Interestingly, Bjerke and coworkers also provided genetic evidence for the existence of at least one additional, yet-unknown, but essential functional entity in pUL34 of HSV-1, besides its known pUL31 binding activity, using a screen based on charged-cluster mutations (1). Further analysis of one of the noncomplementing charged-cluster mutants carrying the defect in the N-terminal domain of pUL34 also revealed a DN activity and suggested a new functional site involved in membrane curvature formation, together with the C-terminal domain of UL31 (34).The genetic analysis of M53 by Tn7-based linker scanning mutagenesis, followed by a cis complementation assay, localized the M50-binding site between aa 112 and 137 within the first of the four conserved regions (CRs) shared among the herpesvirus UL31 homologues (21). This analysis, together with a study we performed for further characterization of pM50/pM53 interaction, revealed that the large C-terminal part of pM53, comprising CR2 to -4, must carry at least one additional, yet-unknown, but essential functional site (21, 38).Here, we screened loss-of-function mutants of the MCMV M53 gene to retrieve M53 alleles with DN activity to localize this new functional domain. Mutants with a very strong inhibitory potential accumulated within CR4 of pM53 close to its C terminus. These CR4 mutants induced a block of capsid export from the nucleus. In addition, we could associate these mutations with the induction of a defect in capsid maturation and/or DNA packaging. These data suggested that pM53 is not only crucial for nuclear egress, but also involved in earlier steps of MCMV morphogenesis.