Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIα-XRCC1 complex. Of these, ligase IIIα-XRCC1 is the most efficient. Moreover, ligase IIIα-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.Herpes simplex virus-1 (HSV-1)² is a large double-stranded DNA virus with a genome of ∼152 kilobase pairs (for reviews, see Refs. 1 and 2). HSV-1 switches between lytic replication in epithelial cells and a state of latency in sensory neurons during which there is no detectable DNA replication (1). Viral DNA replication is mediated by seven essential virus-encoded factors (3–5). Of these, two encode subunits of the viral replicase (for review, see Refs. 6 and 7). The catalytic subunit (UL30) exhibits DNA polymerase (Pol), 3′-5′ proofreading exonuclease, and RNase H activities (8–11). UL30 exists as a heterodimer with the UL42 protein that confers a high degree of processivity on the Pol (11–17).Viral DNA replication is accompanied by vigorous recombination that leads to the formation of large networks of viral DNA replication intermediates (18). The HSV-1 single-strand DNA-binding protein (ICP8) has been shown to play a major role in mediating these recombination reactions (19–21). One role for the high frequency of recombination is to restart DNA replication at sites of fork collapse. Further mechanisms that contribute to genome maintenance are processes that survey and repair damage to the DNA to ensure the availability of a robust replication template. In this regard base excision repair (BER) is essential to remove unusual bases from the DNA and to repair apurinic/apyrimidinic (AP) sites resulting from spontaneous base loss (for review, see Ref. 22). With respect to HSV-1, a recent study showed that viral DNA from infected cultured fibroblasts contains a steady state of 2.8–5.9 AP sites per viral genome equivalent (23). Because AP sites are non-instructional, the failure to repair such sites would terminate viral replication. Indeed, UL30 cannot replicate beyond a model AP site (tetrahydrofuran residue) (23), indicating that the virus must enable a process to repair such lesions. In this regard HSV-1 possesses several enzymes that would safeguard from the accumulation of unusual bases, specifically uracil, and base loss. Hence, HSV-1 encodes a uracil DNA glycosylase (UDG) (UL2) as well as a dUTPase to reduce the pool of dUTP and prevent misincorporation by the viral Pol (24, 25). Moreover, we recently showed that the catalytic subunit of the viral Pol (UL30) exhibits AP and 5′-deoxyribose phosphate (dRP) lyase activities (26). The presence of a virus-encoded UDG and DNA lyase indicates that HSV-1 has the capacity to perform integral steps of BER, specifically for the removal of uracil. Indeed, the excision of uracil may be important for viral replication. Hence, it has been shown that uracil substitutions in the viral origins of replication alters their recognition by the viral initiator protein (27). Moreover, whereas UL2 may be dispensable for viral replication in fibroblast (24), UL2 mutants exhibit reduced neurovirulence and a decreased frequency of reactivation from latency (28). Thus, UDG action in HSV-1 may be important for viral reactivation after quiescence in neuronal cells during which the genome may accumulate uracil as a result of spontaneous deamination of cytosine. In another herpesvirus, cytomegalovirus, the viral UDG was shown to be required for the transition to late-phase DNA replication (29, 30). Consequently, it is possible that BER plays a significant role in various aspects of the herpesvirus life cycle.In mammalian single-nucleotide BER initiated by monofunctional DNA glycosylases, the resulting AP sites are incised hydrolytically at the 5′ side by AP endonuclease (APE), generating a 3′-OH. This is followed by template-directed incorporation of one nucleotide by Pol β to generate a 5′-dRP flap (22, 31, 32). The 5′-dRP residue is subsequently removed by the 5′-dRP lyase activity of Pol β to leave a nick with a 3′-OH and 5′-phosphate that is ligated by DNA ligase I or the physiologically more relevant ligase IIIα-XRCC1 complex (for review, see Refs. 33 and 34). Here we show that the HSV-1 UDG (UL2) and Pol (UL30) cooperate with human APE and human ligase IIIα-XRCC1 complex to perform BER in vitro. This finding has implications on the role of BER in viral genome maintenance during lytic replication and in the emergence of the virus from neuronal latency.     

A Novel Function of Heparan Sulfate in the Regulation of Cell-Cell Fusion

Despite the important contribution of cell-cell fusion in the development and physiology of eukaryotes, little is known about the mechanisms that regulate this process. Our study shows that glycosaminoglycans and more specifically heparan sulfate (HS) expressed on the cell surface and extracellular matrix may act as negative regulator of cell-cell fusion. Using herpes simplex virus type-1 as a tool to enhance cell-cell fusion, we demonstrate that the absence of HS expression on the cell surface results in a significant increase in cell-cell fusion. An identical phenomenon was observed when other viruses or polyethylene glycol was used as fusion enhancer. Cells deficient in HS biosynthesis showed increased activity of two Rho GTPases, RhoA and Cdc42, both of which showed a correlation between increased activity and increased cell-cell fusion. This could serve as a possible explanation as to why HS-deficient cells showed significantly enhanced cell-cell fusion and suggests that HS could regulate fusion via fine tuning of RhoA and Cdc42 activities.Cell-cell fusion is an important physiological process widespread in organisms ranging from yeast to humans (1). It is critical for several biological phenomena including fertilization, placenta formation, skeletal muscle and bone development, tumorigenesis, immune response, and stem cell differentiation (1–9). Defects in cell-cell fusion can lead to serious diseases, such as myotonic dystrophy, centronuclear myopathy, preeclampsia, and osteopetrosis (10–13). Defects in sperm-egg fusion are a major cause of infertility (5). Cell-cell fusion has also been utilized for therapeutic applications, including the generation of monoclonal antibody-producing hybridomas (14) as well as new agents for cancer immunotherapy (15–17).Because of its critical nature, many studies have looked at the mechanism by which cell-cell fusion occurs. Although it can occur in a variety of different biological processes, many of the fusion events share common characteristics (8). For example, tetraspanin proteins function in gamete-, myoblast-, macrophage-, and virus-mediated fusion events (18–21). Although many mediators of cell-cell fusion are known, little is known about the fine-tuning mechanisms that may regulate the membrane fusion process.Viruses have been a useful tool for studying cell-cell fusion since the discovery that they could induce the fusion of somatic cells in vitro (22). Enveloped viruses, like herpes simplex virus type-1 (HSV-1),² use transmembrane viral proteins to mediate fusion with the host cell during entry and spread (23–25). For HSV-1, fusion occurs after the virus has attached to host cells by binding to heparan sulfate (HS) using glycoproteins gB and gC (26). Fusion of the virus envelope with the plasma membrane requires that an additional glycoprotein, gD, binds to one of its receptors, a process that also requires HSV-1 gB, gH, and gL (27–29). During HSV-1-mediated cell-cell fusion, gB, gD, gH, and gL are expressed on the surface of infected cells, allowing them to bind and fuse with surrounding uninfected cells, forming syncytia.Heparan sulfate proteoglycans are ubiquitously expressed cell surface molecules composed of a protein core, commonly syndecan, covalently attached to one or more HS glycosaminoglycan (GAG) side chains via a linker region (30). HS polysaccharide chains are composed of alternating hexuronic acid and d-glucosamine units (30, 31). HS chains undergo extensive modifications during their biosynthesis, including sulfation and epimerization, resulting in a variety of structurally diverse HS chains (30, 32–33). This diversity allows HS to interact with an array of functionally unrelated proteins and participate in various processes, such as the regulation of embryonic development, angiogenesis, blood coagulation, growth factor/cytokine interactions, cell adhesion, and lipid metabolism (30).Much remains to be learned about the cell-cell fusion mechanism and regulation of this phenomenon. The purpose of our study was to examine the effect of HS on cell-cell fusion and how it may function in the fusion mechanism. Using HSV-1 as a tool, we discovered that the absence of HS from the cell surface significantly enhanced the ability of cells to fuse with each other. This effect was also seen independently of HSV-1 in cells that neither expressed HSV-1 glycoproteins nor their receptors. This suggests a novel role for HS as a negative regulator and a fine-tuner of cell-cell fusion events.     

Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB⁻ genome (PrV-gK^gB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.Herpesvirions are complex structures consisting of a nucleoprotein core, capsid, tegument, and envelope. They comprise at least 30 structural proteins (35). Pseudorabies virus (PrV), a member of the Alphaherpesvirinae, is an economically important animal pathogen, causing Aujeszky’s disease in swine. It is also highly pathogenic for most other mammals except higher primates, including humans (28, 45), and a wide range of cultured cells from different species support productive virus replication, reflecting the wide in vivo host range. Envelope glycoproteins play major roles in the early and late interactions between virion and host cell. They are required for virus entry and participate in release of free virions and viral spread by direct cell-to-cell transmission (27, 37). For PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN, have been characterized (20, 27); these glycoproteins are involved in the attachment of virion to host cell (gC and gD), fusion of viral envelope and cellular cytoplasmic membrane (gB, gD, gH, and gL), spread from infected to noninfected cells (gB, gE, gH, gI, gL, and gM), and egress (gC, gE, and gI) (27, 37). Homologs of these glycoproteins are also present in other alphaherpesviruses (37). The gene coding for a potential 11th PrV glycoprotein, gK, has been described recently (3), but the protein and its function have not been identified.The product of the homologous UL53 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) is gK (13, 32). gK was detected in nuclear membranes and in membranes of the endoplasmic reticulum but was not observed in the plasma membrane (14). Also, it did not appear to be present in purified virion preparations (15). The latter result was surprising since earlier studies identified several mutations in HSV-1 gK resulting in syncytium-inducing phenotypes (7, 14), which indicates participation of gK in membrane fusion events during HSV-1 infection. Moreover, HSV-1 mutants in gK exhibited a delayed entry into noncomplementing cells, which is difficult to reconcile with absence of gK from virions (31). Mutants deficient for gK expression have been isolated and investigated by different groups (16, 17). Mutant F-gKβ carries a lacZ gene insertion in the HSV-1 strain F gK gene, which interrupts the ORF after codon 112 (16). In mutant ΔgK, derived from HSV-1 KOS, almost all of the UL53 gene was deleted (17). Both mutants formed small plaques on Vero cells, and virus yield was reduced to an extent which varied with the different confluencies of the infected cells, cell types, and mutants used for infection. However, both HSV-1 gK mutants showed a defect in efficient translocation of virions from the cytoplasm to the extracellular space, and only a few enveloped virions were present in the extracellular space after infection of Vero cells (16, 17). The authors therefore suggested that HSV-1 gK plays a role in virion transport during egress.Different routes of final envelopment and egress of alphaherpesvirions are discussed. It has been suggested that HSV-1 nucleocapsids acquire their envelope at the inner nuclear membrane and are transported as enveloped particles through the endoplasmic reticulum to the Golgi stacks, where glycoproteins are modified in situ during transport (5, 6, 19, 39), although other potential egress pathways cannot be excluded (4). In contrast, maturation of varicella-zoster virus and PrV involves primary envelopment at the nuclear membrane, followed by release of nucleocapsids into the cytoplasm and secondary envelopment in the trans-Golgi area (10, 12, 43). Final egress of virions appears to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. The possibility of different routes of virion egress is supported by studies of other proteins involved in egress, e.g., the UL20 proteins of HSV-1 and PrV and the PrV UL3.5 protein, which lacks a homolog in the HSV-1 genome (1, 8, 9). In UL20-negative HSV-1, virions accumulated in the perinuclear cisterna of Vero cells (1), while PrV UL20⁻ virions accumulated and were retained in cytoplasmic vesicles (9). PrV UL3.5 is important for budding of nucleocapsids into Golgi-derived vesicles during secondary envelopment (8). Thus, there appear to be profound differences in the egress pathways. Since HSV-1 gK was also implicated in egress, we were interested in identifying the PrV homolog and analyzing its function.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]