The flavivirus nonstructural glycoprotein NS1 is highly conserved and contains two N-linked glycosylation sites which are both utilized for addition of oligosaccharides during replication in cell culture. NS1 has been shown to contain epitopes for protective antibodies; however, its roles in virus replication and pathogenesis remain unknown. To study the function of NS1 during yellow fever virus replication, six mutant viruses which lack either one or both glycosylation sites and another one containing silent mutations at both sites were generated by site-directed mutagenesis. Mutants lacking the second glycosylation site and those bearing silent mutations were similar to the parental virus in their cell culture properties. Ablation of the first or both glycosylation sites generated mutants exhibiting small plaque phenotypes, decreased virus yields, reduced cytopathic effects, impaired NS1 secretion, and depressed RNA accumulation. In addition, mutants lacking the first or both glycosylation sites exhibited significant reduction in mouse neurovirulence after intracerebral inoculation. These defects appear to result from the lack of N-linked glycans rather than the introduction of deleterious amino acid substitutions or disruption of cis-acting RNA elements important for RNA replication. These results suggest an important role for NS1 in flavivirus RNA replication and pathogenesis.

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IVdeficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection. Herpes simplex virus type I (HSV-1)2 has a 152-kb linear double-stranded genome with direct repeats, a sequences, presented at the genomic termini (1). Once the genomic DNA is delivered through the nuclear pores, it rapidly becomes converted to an endless state (2-4). The endless genomes may be circular molecules, products of rolling circle replication, and/or concatemers formed by recombination or intermolecular ligation. It has been a commonly held opinion that circular genomes are replicated from the viral origins of replication, oriS and oriL, by theta type replication and that later a switch into a rolling circle mode of replication will take place (1). As a result, the products of viral DNA synthesis appear as linear concatemers, which are the immediate precursors for the cleavage packaging process. This view has recently received strong support by the observation that a genetically modified HSV-1 genome undergoes fusion of the termini rapidly upon infection and in the presence of inhibitors of viral DNA synthesis (4). Alternatively, it has been proposed that circularization does not take place during the productive phase of wild-type HSV-1 infection (5, 6). Instead, the linear genomes are substrates for replication, and concatemers are formed later by recombination. Circular molecules, on the other hand, are proposed to be unique to latently infected neurons.To provide some insights into the issues raised above, it would be beneficial to identify the enzymes required for replication and recombination of viral genomes. HSV-1 encodes seven gene products directly involved in DNA synthe...

The flavivirus NS1 protein is a highly conserved nonstructural glycoprotein that is capable of eliciting protective immunity. NS1 homodimers are secreted from virus-infected mammalian cells, but the protein is also present at the plasma membrane and in the lumen of intracellular vesicles. Based on these properties, it has been speculated that NS1 may function in virus maturation or release. To gain further insight into NS1 function, we used clustered charged-amino-acid-to-alanine mutagenesis to create 28 clustered substitutions in the NS1 protein of yellow fever virus. To screen for conditional mutations, full-length RNAs containing each mutation were assayed for plaque formation at 32 and 39؇C after RNA transfection. We found that 9 mutations were lethal, 18 allowed plaque formation at both temperatures, and 1, ts25, was strongly heat sensitive and was unable to form plaques at 39؇C. Lethal mutations clustered in the amino-terminal half of NS1, whereas those leading to impaired replication relative to the parent were distributed throughout the protein. High-multiplicity infections at 39؇C demonstrated that ts25 was defective for RNA accumulation, leading to depressed viral protein synthesis and delayed virus production. Although ts25 secreted less NS1 than did the parent, temperature shift experiments failed to demonstrate any temperature-dependent differences in polyprotein processing, NS1 stability and secretion, or release of infectious virus. The ts lesion of ts25 was shown to be due to a single alanine substitution for Arg-299, a residue which is conserved among flaviviruses. These results argue that NS1 plays an essential but as yet undefined role in flavivirus RNA amplification.

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the singlestranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.Herpesviruses are found in all animals from molluscs to man. During evolution, the viruses have become tightly associated and co-evolved with their hosts. They seem to cross species borders only by accident, and in such rare instances, they may cause unexpected and severe disease. Herpesviruses have an unusual lifestyle; they cause lytic infection in cells, leading to efficient production of new infectious virus particles, and they also establish latent infections in either non-dividing neuronal cells or cycling cells of the immune system. The latent state is characterized by expression of a very limited set of genes to ascertain maintenance of virus chromosomes and to escape recognition by the immune system. The mechanisms for establishing latency appear to differ considerably for different herpesviruses. In contrast, mechanisms for replication of virus DNA during lytic infection and subsequent formation of infectious particles seem to be evolutionarily conserved. There is one notable exception; the mechanism for recognition of origins of DNA replication and initiation of DNA synthesis differs between the herpesvirus families.Humans can be infected by eight different herpesviruses. Herpes simplex viruses I and II and varicella zoster virus are alphaherpesviruses. Cytomegalovirus and the roseoloviruses, human herpesviruses 6 and 7, are classified as betaherpesviruses. Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus belong to the gammaherpesvirus subfamily.In this minireview, we discuss recent developments in replication, recombination, and repair of herpes simplex virus DNA. We will take as our starting point a previous minireview in the Journal of Biological Chemistry that provides an insightful and accurate description of basic mechanisms and components of the herpes simplex virus replication machinery (1). Noteworthy new developments have been (i) the presentation of three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein (ssDNA) 2 ; (ii) the recons...