Clinical resistance to antiviral drugs requies that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants ofherpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and tanslation ofmutant tkgenes, incuding constructs in which reporter epitopes could be expressed only if frameshiffing occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading fram cloning failed to detect alterations in tktrspts that could account for the synthesis offull-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frmeshlft during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.Herpes simplex virus (HSV) is an important human pathogen, especially in patients with AIDS. A major advance in antiviral therapy has been the use of acyclovir to treat HSV infections, but acyclovir resistance is a problem of increasing clinical significance in immunocompromised patients (1). Clinical resistance implies that HSV can mutate to evade drug therapy yet retain pathogenicity. Because the sensitivity of HSV to acyclovir is due largely to the viral thymidine kinase (TK), which activates the drug (2), HSV tk mutations can confer acyclovir resistance (3). Indeed, there have been numerous cases ofpatients who suffered severe HSV disease despite acyclovir therapy and shed acyclovir-resistant viruses that were reported to express no detectable TK activity and/or full-length TK polypeptides (4-13).Although TK is not essential for viral replication in cell culture, it is important for viral pathogenesis in animal models. In particular, TK-mutants fail to reactivate from latent infections of mouse sensory ganglia (14-16), due to the tk mutation (16). Thus, the association of virus that appears TK-with severe HSV disease has been puzzling. One possible resolution of this paradox is that the clinical isolates expressed low levels of TK that were not detected. There are HSV mutants that are severely impaired for TK activity and thus acyclovir-resistant...

Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.DNA polymerase (Pol) is the pivotal enzyme involved in DNA replication and plays a central role in controlling the accuracy of DNA replication. Amino acid sequence alignment of various Pols reveals that all Pols contain conserved residues distributed within several regions of the Pol domain. Previous studies demonstrated that these conserved residues play an important role in the polymerization reaction (reviewed in reference 38 and references therein). These conserved residues interact with incoming deoxyribonucleotides (deoxynucleoside triphosphates [dNTPs]) and the primer-template DNA. Therefore, mutations within these residues could result in altered polymerase activity and/or replication fidelity.Herpes simplex virus (HSV) has proven to be a good model for genetic, molecular biological, and biochemical studies of Pol, since it can be manipulated to contain desired mutations for further characterization regardless of whether the mutated Pol is lethal for viral replication (reviewed in reference 11 and references therein). Examination of the HSV-1 Pol also reveals seven conserved regions within the carboxyl-terminal half of the Pol, which has been defined as the polymerase domain (Fig. 1). Indeed, mutations within most of these conserved regions confer altered sensitivities to certain nucleoside and pyrophosphate analog antiviral drugs, indicating that they are involved in the interactions with dNTPs and DNA (11).Since only a few region VI mutants of HSV have been described (4, 5, 34), and the effects of these mutations on replication fidelity have not been characterized, we constructed recombinant HSV-1 viruses containing a region VI mutation (L774F) for characterizing the effects of this mutatio...

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.A LL microbes with chromosomes composed of DNA, However, recently reported data can be used to fill this gap. The wild-type KOS strain of herpes simplex virus and for which both spontaneous mutation rates and mutational spectra have been determined, display type 1 (HSV-1) cannot form plaques in the presence of various nucleoside analogs, while thymidine kinase (tk) a mutation rate in the laboratory of ‫300.0ف‬ per genome replication, with a range of 0.0018-0.0046 and boundary mutants defective in the synthesis of thymidine kinase can do so. Ganciclovir is a guanosine analog that bevalues experimentally indistinguishable from 0.003 (Drake et al. 1998; Grogan et al. 2001). The DNA-based comes strongly cytotoxic when phosphorylated. Using ganciclovir as the selective agent provided a HSV-1 tk Ϫ microbes that support this value are phage M13 with single-stranded DNA and a temperate mode of infection; mutant frequency of 6 ϫ 10 Ϫ5 and a mutation spectrum (Lu et al. 2002). To calculate a genomic mutation rate, phage with double-stranded DNA and alternating bethis value must first be corrected for tk mutations that tween lysogenic and lytic modes of infection; phages T2were not detected. A preferred way to do this (Drake and T4 with double-stranded DNA and a strictly lytic 1991) is to assume that indels (insertions and deletions mode of infection; the eubacterium Escherichia coli growof base pairs) within the coding sequence are detected ing optimally at ‫73ف‬Њ; the archaeon Sulfolobus acidocaldwith high efficiency and that the total number of BPSs arius growing optimally at ‫08ف‬Њ; the yeast Saccharomyces can be estimated from the number that produce intercerevisiae; and the filamentous fungus Neurospora crassa.nal stop codons (UAG, UGA, and UAA), which are These organisms vary by nearly four orders of magniusually null mutations; this is done by multiplying by 64/3 tude in genome size, and the average gene or base-pair the number of chain-terminating codons produced by mutation rate varies reciprocally by the same amount.BPSs. In the present experiment, the mutational specAnother generality holds in most but not all DNAtrum comprised 67 mutations of which 45 were indels and based organisms: on average, the majority of mutations 5 were chain-terminating BPSs; this proportion (5/22 ϭ are base-pair substitutions (BPSs) (Grogan et al. 2001). 23%) of BPSs that produce chain-terminating codons The exceptions are thermophiles, which appear to have falls in the center of the range (5-50%, average 20%) lower rates of BPSs than do mesophiles (Grogan et al.found for DNA-based microbes (Grogan et al. 2001(Grogan et al. ). 2001 Friedman et al. 2004), perhaps because the averThe correcte...

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.Herpes simplex virus type 1 (HSV-1) carries out gene expression, DNA replication, and DNA encapsidation in globular nuclear domains designated replication compartments (53, 55). These domains contain the essential viral DNA replication proteins (the origin-binding protein, the single-stranded-DNAbinding protein, the helicase-primase subunits, and the polymerase subunits [34,36,55]) and are usually visualized by antibodies either against ICP8, the single-stranded-DNA-binding protein, or UL42, the polymerase processivity subunit. The formation of replication compartments is mediated in part by interactions with nuclear structures called ND10 (nuclear domains 10), promyelocytic leukemia bodies, or PODs (17). The function of ND10 has not yet been defined for cellular or viral growth. Proteins found in ND10 have been associated with the control of cellular growth, cell cycle regulation, transcription, and apoptosis (11,12,24,27,46,71). In the case of the herpesviruses, viral DNA is deposited at ND10 and immediateearly transcripts can be detected at sites adjacent to ND10 (42). Furthermore, replication compartments formed after transfection with the seven essential HSV-1 replication proteins localize adjacent to ND10 (36, 74).ND10 are dynamic structures which are disrupted during mitosis and respond to environmental stimuli including interferon treatment, heat shock, treatment with heavy metals, and viral infection (44,64,65). The most extensively studied ND10 protein, PML, is expressed as a fusion with retinoic acid receptor ␣ in individuals with acute promyelocytic leukemia (31,56). In this disease, disrupti...

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14-to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.Replicative DNA polymerases are highly processive enzymes that synthesize long stretches of DNA without dissociating from the template. Many replicative polymerases depend on accessory proteins called processivity factors, which confer processivity on their cognate polymerases by slowing their dissociation from DNA. Among these are the "sliding clamp" processivity factors (18), exemplified by proliferating cell nuclear antigen (PCNA), the processivity factor for eukaryotic replicative DNA polymerases. All sliding clamps adopt a common structure, in which a dimer or trimer of the sliding clamp protein forms a ring that encircles DNA (20,21,23,28). The association of the sliding clamps with DNA requires the activity of accessory proteins known as clamp loaders (19), which open the clamp ring and reclose it on DNA in an ATP-dependent reaction. The sliding clamp then can tether the catalytic polymerase core to DNA, thus ensuring processivity without impeding the movement of the polymerase.The DNA polymerase that replicates herpes simplex virus (HSV) is a heterodimer of a 137-kDa catalytic subunit (UL30, also called Pol) and a 60-kDa processivity subunit, UL42 (15,24,25). The extreme C terminus of Pol is necessary for interaction with UL42, long-chain DNA synthesis, and viral re...