X-Linked Agammaglobulinemia Patients Are Not Infected with Epstein-Barr Virus: Implications for the Biology of the Virus

Epstein-Barr virus (EBV) infects both B lymphocytes and squamous epithelial cells in vitro, but the cell type(s) required to establish primary and persistent infection in vivo has not been definitively elucidated. The aim of this study was to investigate a group of individuals who lack mature B lymphocytes due to the rare heritable disorder X-linked agammaglobulinemia in order to determine the role of the B cell in the infection process. The results show that none of these individuals harbored EBV in their blood or throat washings. Furthermore, no EBV-specific memory cytotoxic T lymphocytes were found, suggesting that they had not undergone infection in the past. In contrast, 50% of individuals were found to carry human herpesvirus 6, showing that they are infectible by another lymphotropic herpesvirus. These results add weight to the theory that B lymphocytes, and not oropharyngeal epithelial cells, may be required for primary infection with EBV.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

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.     

Tyrosine 112 of Latent Membrane Protein 2A Is Essential for Protein Tyrosine Kinase Loading and Regulation of Epstein-Barr Virus Latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of C. neoformans is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In C. neoformans we identified four predicted endochitinases, CHI2, CHI21, CHI22, and CHI4, and a predicted exochitinase, hexosaminidase, HEX1. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only N-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of C. neoformans during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised individuals. The incidence of cryptococcosis continues to rise in direct proportion to the spread of the human immunodeficiency virus (for review, see Casadevall and Perfect [7]). It is estimated that up to 13% of AIDS patients in the United States will develop life-threatening cryptococcal meningitis, and in some parts of Africa this estimate increases to 40% (7). Current antifungal therapies for treatment of cryptococcosis are inadequate. Amphotericin B, which is believed to interact with membrane sterols (ergosterol) to produce an aggregate that forms a transmembrane channel is effective, but toxic (50, 62). Fluconazole inhibits cytochrome P-450-dependent 14α-sterol demethylase, which leads to the depletion of ergosterol and the accumulation of sterol precursors and results in the formation of a plasma membrane with altered structure and function. It is fungistatic and has high relapse rates (18, 41, 42, 50, 62). Flucytosine can be toxic and resistance occurs frequently (9, 41, 42, 50, 62). The newest class of antifungals to emerge is the echinocandins that targets an essential fungal enzyme required for the synthesis of a β-(1,3)-glucan in the fungal cell wall (17, 34). In addition, the echinocandins have been shown to be safe and effective for treatment of specific fungal infections, including candidiasis and aspergillosis caused by Candida albicans and Aspergillus fumigatus, respectively (23, 59). However, even though C. neoformans possesses the target enzyme β-(1,3)-glucan synthase and in vitro assays have shown the enzyme''s activity to be inhibited by the echinocandin caspofungin (34), C. neoformans still exhibits resistance to this class of drugs (26).Because fungi are eukaryotes and share many biochemical processes with their host, antifungal drug design has been problematic. The cell wall is a prominent structure that differentiates fungi from mammalian host cells. For all fungi, this organelle is essential and provides structure as well as integrity; thus, the cell wall components or their biosynthetic pathways make attractive drug targets. In addition, the cell wall of C. neoformans is associated with a variety of known virulence factors that are important for host-pathogen interactions, and it contains polymers including chitin and chitosan that are necessary for the viability of C. neoformans. The first virulence factor that a host cell encounters is the polysaccharide capsule. The capsule attachment to the outer portion of the cell wall requires α-(1-3)-glucan (15, 46). Another cell wall associated virulence factor is the melanin pigment (61) that is produced by two laccase proteins, Lac1 and Lac2 (38, 44). Lac1 is responsible for generating the majority of melanin and is localized to the cell wall (38, 63, 69). Chitin and chitosan are essential components of the cell wall that have been shown to contribute to the overall strength and integrity of the cell wall (4, 5). The essentiality of the chitin component and the lack of it being present in host cells make chitin and its biosynthetic components attractive targets for drug design.Chitin is one of the most abundant polymers found in nature (1, 12). It is a linear polymer of β-(1,4)-linked N-acetylglucosamine (GlcNAc), and in fungi it is formed from cytoplasmic pools of UDP-GlcNAc. C. neoformans has eight predicted chitin synthases and three putative chitin synthase regulators for synthesis of chitin polymers. Mutational analysis indicate that two chitin synthases, Chs4 and Chs5, produce the majority of vegetative chitin, and one, Chs3, produces the majority of chitin that is converted to chitosan during vegetative growth (5). Chitosan, the deacetylated version of chitin, is produced by chitin deacetylases (EC 3.5.1.41) that remove acetyl groups from nascent chitin polymers. In C. neoformans the chitin produced by Chs3 and the chitin synthase regulator, Csr2, is deacetylated to chitosan by up to three chitin deacetylases (Cda1, Cda2, and Cda3) (4, 5). Strains of C. neoformans lacking either CHS3 or CSR2 have significantly reduced chitosan levels and are sensitive to a variety of cell wall inhibitors (5). Similarly, strains lacking all three chitin deacetylases are unable to convert chitin to chitosan and are sensitive to cell wall inhibitors (4). This indicates that chitosan is essential for the proper maintenance of cell wall integrity in C. neoformans and Chs3, Csr2, and the chitin deacetylases contribute to its formation (4, 5). Chitosan polymers of other fungi have been reported to possess various degrees of deacetylation (57). Chitin and chitosan are located throughout the lateral cell wall and bud neck regions of C. neoformans (4). During growth cellular chitin and chitosan need to be continuously remodeled, presumably through the enzymatic digestion of chitin and chitosan polymers by chitinases and or chitosanases.Chitinases (EC 3.2.1.14) are enzymes that hydrolyze the β-(1-4) linkages in polymers of chitin. Besides being in fungi, these enzymes occur in a wide variety of organisms, including viruses, bacteria, plants, and animals (1, 12). There are two major categories of chitinases: endochitinases and exochitinases. Generally, the endochitinases cleave chitin chains internally to generate low-molecular-mass multimers of GlcNAc. In contrast, the exochitinases are divided into two subcategories: chitobiosidases (EC 3.2.1.29) release diacetylchitobiose from the nonreducing end of chitin chains, and β-(1,4)-N-acetylhexosaminidases (EC 3.2.1.52) release GlcNAc from the nonreducing end of chitin oligosaccharides; both types are usually processive (12). Fungal chitosanases (EC 3.2.1.132) are less understood. They have been found in Aspergillus spp. and Gongronella sp. strain JG. Although these chitosanases have been shown to degrade chitosan, their in vitro physiological relevance has not been elucidated (8, 60).In other fungal systems chitinases are known to be involved in cell separation, hyphal growth and branching, development of reproductive structures, spore germination, and autolysis (1, 12). In the nonpathogenic model yeast Saccharomyces cerevisiae two chitinases, Cts1p and Cts2p, function independently in bud separation and spore formation, respectively (25, 27). Cts1p is the only chitinase expressed during vegetative growth, and strains lacking this enzyme display incomplete cell separation (27) that can lead to pseudohyphalike growth (25). The synthesis of the spore wall is adversely affected by the deletion of CTS2 and affects the ability of the yeast to form mature asci (19).C. neoformans reproduces predominantly by budding, but also has a defined sexual cycle that culminates in the production of basidiospores. Both the yeast and the spore forms are thought to be infectious particles (7). C. neoformans typically colonizes the lungs of a immunocompromised host, from where it can disseminate to the central nervous system (7). As such, reproduction by budding has been shown to occur within host macrophages and dendritic cells (3, 28). Because fungal chitinases in other systems such as S. cerevisiae and C. albicans have been shown to be necessary for the completion of cell division (11, 27), understanding the biosynthesis and activity of chitinases could determine whether interfering with chitinase activity would impair the ability of C. neoformans to reproduce.We hypothesized that the chitinases in C. neoformans would be involved in growth and, like the chitinases in S. cerevisiae and C. albicans, that they would degrade specific chitin during either bud separation, hyphal growth, or sporulation. In the present study we utilized a homology-based search to identify five potential chitinases in C. neoformans, the four endochitinases CHI2, CHI21, CHI22, and CHI4 and one exochitinase, HEX1. Using a panel of chitinase deletion strains we discovered that the chitinases are dispensable for “normal” vegetative growth but were necessary during development of the sexual phase of C. neoformans.     

黄瓜花叶病毒2b蛋白C末端无抑制局部RNA沉默的活性

目的:黄瓜花叶病毒 (Cucumber mosaic virus,CMV) 编码的2b蛋白具有RNA沉默抑制子的功能,其C末端氨基酸序列非常保守。为了明确2b蛋白C末端保守序列在RNA沉默抑制中的作用,构建了CMV Q株系野生型2b及其C末端缺失突变体2b^delC的植物瞬时表达载体。通过农杆菌共渗滤法对野生型2b及其C末端突变体的沉默抑制子活性进行了分析。结果与结论:烟草接种叶片中野生型2b及其C末端突变体的Western blot检测表明,野生型2b蛋白与其C末端突变体在植物中积累水平变化不大,说明2b蛋白C末端氨基酸残基在维持2b蛋白在植物细胞中的稳定性方面无作用。在整株、细胞和分子水平上分别比较了野生型2b及其突变体2b^delC对共表达GFP的表达量影响,结果表明在所有的测定结果中二者均无明显地差异,说明2b蛋白C末端94-111位氨基酸在抑制局部RNA沉默上无生物学活性,讨论推测C末端应不存在与小RNA结合的结构域。     

Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG^Met→GCC^Ala mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.     

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2, 7, 34, 36, 39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11, 13, 15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm and transiently localizes to nucleoli (23). In the absence of other viral proteins, UL24 accumulates in the Golgi apparatus and in the nucleus, where it usually exhibits a diffuse staining pattern, but in a minority of cells it is detected in nucleoli (3).During infection, the formation of the viral replication compartments in the nucleus and the action of several viral proteins result in a remodeling of the nucleus. Chromatin is marginalized (29, 40), promyelocytic leukemia bodies are dispersed (26, 27), and the nuclear lamina is disrupted (33, 37). HSV-1 infection also affects the nucleolus, a prominent nuclear substructure implicated in the synthesis of rRNA, cell cycle regulation, and nucleocytoplasmic shuttling (5). Nucleoli become elongated following infection, and the synthesis of mature rRNA is reduced (4, 38, 42). Several HSV-1 proteins have been shown to localize to, or associate with, the nucleolus (12). The viral protein VP22 associates with the nucleolus and with dispersed nucleolin in HSV-1-infected cells (22), and RL1, US11, and ICP0 have also been shown to localize to nucleoli (24, 30, 35). Previously we showed that nucleolin is dispersed throughout the nucleus upon HSV-1 infection and that UL24 is involved in this nuclear modification (23). We further found that the N-terminal portion of UL24 is sufficient to induce the redistribution of nucleolin in the absence of other viral proteins (3).In this study, we sought to test the hypothesis that the endonuclease motif, which is made up of some of the most highly conserved residues in UL24, is important for the dispersal of nucleolin. A panel of substitution mutations in UL24 was generated, and the impact on the function of UL24 was assessed.     

Glycoproteins E and I of Marek's Disease Virus Serotype 1 Are Essential for Virus Growth in Cultured Cells

The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20DeltagE), gI (20DeltagI), or both gE and gI (20DeltagEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088-11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20DeltagE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20DeltagI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20DeltagEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae.     

African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.