Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important.

The classical recessive mouse mutant, Purkinje cell degeneration (pcd), exhibits adult-onset degeneration of cerebellar Purkinje neurons, retinal photoreceptors, olfactory bulb mitral neurons, and selected thalamic neurons, and has defective spermatogenesis. Here we identify Nna1 as the gene mutated in the original pcd and two additional pcd alleles (pcd2J and pcd3J). Nna1 encodes a putative nuclear protein containing a zinc carboxypeptidase domain initially identified by its induction in spinal motor neurons during axonal regeneration. The present study suggests an unexpected molecular link between neuronal degeneration and regeneration, and its results have potential implications for neurodegenerative diseases and male infertility.

The NS5A protein of hepatitis C virus (HCV) plays an important but undefined role in viral RNA replication. NS5A has been proposed to be a three-domain protein, and the crystal structure of the well-conserved amino-terminal domain I has been determined. The remaining two domains of NS5A, designated domains II and III, and their corresponding interdomain regions are poorly understood. We have conducted a detailed mutagenesis analysis of NS5A domains II and III using the genotype 1b HCV replicon system. The majority of the mutants containing 15 small (8-to 15-amino-acid) deletions analyzed were capable of efficient RNA replication. Only five deletion mutations yielded lethal phenotypes, and these were colinear, spanning a 56-amino-acid region within domain II. This region was further analyzed by combining triple and single alanine scanning mutagenesis to identify individual residues required for RNA replication. Based upon this analysis, 23 amino acids were identified that were found to be essential. In addition, two residues were identified that yielded a small colony phenotype while possessing only a moderate defect in RNA replication. These results indicate that the entire domain III region and large portions of domain II of the NS5A protein are not required for the function of NS5A in HCV RNA replication.

In 1993, two groups showed that X-linked agammaglobulinemia (XLA) was due to mutations in a tyrosine kinase now called Btk. Most laboratories have been able to detect mutations in Btk in 80%-90% of males with presumed XLA. The remaining patients may have mutations in Btk that are difficult to identify, or they may have defects that are phenotypically similar to XLA but genotypically different. We analyzed 101 families in which affected males were diagnosed as having XLA. Mutations in Btk were identified in 38 of 40 families with more than one affected family member and in 56 of 61 families with sporadic disease. Excluding the patients in whom the marked decrease in B cell numbers characteristic of XLA could not be confirmed by immunofluorescence studies, mutations in Btk were identified in 43 of 46 patients with presumed sporadic XLA. Two of the three remaining patients had defects in other genes required for normal B cell development, and the third patient was unlikely to have XLA, on the basis of results of extensive Btk analysis. Our techniques were unable to identify a mutation in Btk in one male with both a family history and laboratory findings suggestive of XLA. DNA samples from 41 of 49 of the mothers of males with sporadic disease and proven mutations in Btk were positive for the mutation found in their son. In the other 8 families, the mutation appeared to arise in the maternal germ line. In 20 families, haplotype analysis showed that the new mutation originated in the maternal grandfather or great-grandfather. These studies indicate that 90%-95% of males with presumed XLA have mutations in Btk. The other patients are likely to have defects in other genes.

The ␣9 acetylcholine receptor (␣9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse ␣9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of ␣9 AChR. However, we observed previously unidentified dynamic changes in ␣9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of ␣9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of ␣9 AChR expression in the basal turn. In the utricle, we observed high levels of ␣9 AChR expression in the striolar region during early neonatal development and high levels of ␣9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and ␣9 AChR expression showed that dynamic expression of ␣9 AChR in developing hair cells was independent of efferent contacts. We propose that ␣9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.

bThe hepatitis C virus NS5A protein is essential for RNA replication and virion assembly. NS5A is phosphorylated on multiple residues during infections, but these sites remain uncharacterized. Here we identify serine 222 of genotype 2a NS5A as a phosphorylation site that functions as a negative regulator of RNA replication. This site is a component of the hyperphosphorylated form of NS5A, which is in good agreement with previous observations that hyperphosphorylation negatively affects replication. Hepatitis C virus (HCV) is a human pathogen of global impact, with as many as 170 million chronically infected (1). Longterm infection results in progressive liver damage, including fibrosis, cirrhosis, and, oftentimes, hepatocellular carcinoma. HCV is a member of the Flaviviridae family of enveloped, positive-sense, single-stranded RNA viruses (reviewed in reference 2). The noncapped, nonpolyadenylated, 9.6-kb viral genome encodes a single internal ribosome entry site (IRES)-directed open reading frame flanked by highly structured 5= and 3= nontranslated regions. Translation yields a polyprotein of approximately 3,000 amino acids that undergoes a complex series of co-and posttranslational proteolytic events catalyzed by viral and host proteases, resulting in the production of 10 mature HCV proteins. These include the structural proteins that are involved in the production of progeny virions (C, E1, E2, p7) and the nonstructural replication proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). There is considerable overlap in functionality between these groups, with a number of nonstructural proteins serving essential functions for both RNA replication and virion assembly, including the NS5A protein (3-11).NS5A is a large, three-domain, hydrophilic phosphoprotein that exists in two distinct forms: a hypophosphorylated form of roughly 56 kDa and a hyperphosphorylated form of 58 kDa (12, 13). It is not known what residues in NS5A are phosphorylated or how various phosphorylation events contribute to the presence of the two phospho-forms (see reference 14 for a concise review). Evidence from mutagenesis suggests that hypophosphorylation primarily targets serine residues in domains II and III, whereas hyperphosphorylation sites cluster in and around domain I, LCSI (low complexity sequence block I), and domain II (12,(15)(16)(17)(18). The function of NS5A phosphorylation in the viral life cycle is unknown, but a general theme has emerged in which hypophosphorylation is required for RNA replication, and hyperphosphorylation is a negative regulator of this process. Phosphorylation may also play a role in the function of NS5A in virion assembly, and it could regulate the interface between replication and assembly events (11). A number of phosphorylation sites have been identified in NS5A using different overexpression systems, but it is unknown in some cases if these modifications are actually used in the virus life cycle or what their function(s) might be (16-22).As no phosphorylation sites have yet been definitively mapped in NS5A i...

The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions. IMPORTANCEInformation about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes. Hepatitis C virus (HCV) is an enveloped, positive-sense, singlestranded RNA virus belonging to Hepacivirus genus within the Flaviviridae family. The HCV open reading frame (ORF) encodes a polyprotein of ϳ3,011 amino acids (aa), which is cleaved co-and posttranslationally by viral and host proteases into 10 viral proteins: the core; the envelope glycoproteins E1 and E2; the viroporin p7; and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Among the NS proteins, NS2 with the protease activity is required for virion assembly; NS3 is a multifunctional protein with serine protease activity at the N-terminal region, while the C-terminal portion of the protein holds helicase/ nucleotide triphosphatase (NTPase) activity; NS4A acts as a cofactor for the NS3 serine protease; NS4B serves the scaffold for the HCV replication complex (RC) by inducing the formation of the membranous web (MW); NS5A is a phosphoprotein that plays a key role in HCV RNA replication and viral assembly processes; and NS5B is the RNA-dependent RNA polymerase (1-5).Like all other positive-strand RNA viruses, HCV RNA replicates in close association with cellular membranes. HCV alters the en...

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