Herpes Simplex Virus Type 1 Replication Research Articles

Evaluation of Antiviral Activity of Compounds Isolated fromRanunculus sieboldiiandRanunculus sceleratus

Nineteen compounds isolated from Ranunculus sieboldii and Ranunculus sceleratus were tested for inhibitory effects on hepatitis B virus (HBV) and Herpes simplex virus type-1 (HSV-1). The results showed that apigenin 4'- O- alpha-rhamnopyranoside, apigenin 7- O- beta-glucopyranosyl-4'- O- alpha-rhamnopyranoside, tricin 7- O- beta-glucopyranoside, tricin, and isoscopoletin possessed inhibitory activity against HBV replication. Protocatechuyl aldehyde exhibited an inhibiting activity on HSV-1 replication. It is therefore suggested that further investigations on these bioactive compounds might be needed to discover and develop new antiviral agents.

Anti-HSV-1 activity of Euclea natalensis

The inhibitory effect of crude extracts of Euclea natalensis A. DC. and a naphthoquinone, diospyrin, isolated from this species, was investigated against herpes simplex virus Type 1 (HSV-1) for activity by in vitro studies on primary vervet monkey kidney (VK) cells. Cell toxicity was monitored by determining the effect of the extracts and diospyrin on the monolayers of VK cells. The dose of the plant samples that inhibited 50% cell growth (ID 50 ) after the incubation period was 0.1 mg ml -1 and 0.2 mg ml -1 for acetone and water extracts, respectively. Diospyrin exhibited an ID 50 of 0.02 mg ml -1 on VK cells. The water extract from the roots of the plant was the least toxic to cell cultures and inhibited the replication of HSV-1 moderately at a concentration of 0.2 mg ml –1 . Diospyrin had no inhibitory effect against the virus. However, acetone extracts inhibited the replication of the virus at concentrations ranging from 0.1 mg ml –1 to 0.02 mg ml –1 , as shown by the reduction of virus- induced cytopathogenic effects and the protection of cells in a cell viability assay

Beta interferon and gamma interferon synergize to block viral DNA and virion synthesis in herpes simplex virus-infected cells

The capacity of herpes simplex virus type 1 (HSV-1) to replicate in vitro decreases tremendously when animal cell cultures are exposed to ligands of both the alpha/beta interferon (IFN-alpha/beta) receptor and IFN-gamma receptor prior to inoculation with low m.o.i.s of HSV-1. However, the available evidence provides no insight into the possible mechanisms by which co-activation of the IFN-alpha/beta- and IFN-gamma-signalling pathways produces this effect. Therefore, it has not been possible to differentiate between whether these observations represent an important in vitro model of host immunological suppression of HSV-1 infection or an irrelevant laboratory phenomenon. Therefore, the current study was initiated to determine whether co-activation of the host cell's IFN-alpha/beta and IFN-gamma pathways either (i) induced death of HSV-1-infected cells such that virus replication was unable to occur; or (ii) disrupted one or more steps in the process of HSV-1 replication. To this end, multiple steps in HSV-1 infection were compared in populations of Vero cells infected with HSV-1 strain KOS (m.o.i. of 2.5) and exposed to ligands of the IFN-alpha/beta receptor, the IFN-gamma receptor or both. The results demonstrated that IFN-beta and IFN-gamma interact in a synergistic manner to block the efficient synthesis of viral DNA and nucleocapsid formation in HSV-1-infected cells and do so without compromising host-cell viability. It was inferred that IFN-mediated suppression of HSV-1 replication may be a central mechanism by which the host immune system limits the spread of HSV-1 infection in vivo.

Herpes simplex virus type 1 propagation in HeLa cells interrupted by Nelumbo nucifera

Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, NN-B-5 was identified from seeds of N. nucifera. NN-B-5 significantly blocked HSV-1 multiplication in HeLa cells without apparent cytotoxicity. To elucidate the point in HSV-1 replication where arrest occurred, a set of key regulatory events leading to the viral multiplication was examined, including HSV-1 DNA synthesis and viral immediate early gene expressions. Data from polymerase chain reaction and Southern blotting showed that there were impairments of HSV-1 DNA replication in HeLa cells treated with NN-B-5. Results indicated that the production and mRNA transcription of infected cell protein (ICP) 0 and ICP4 were decreased in NN-B-5 treated HeLa cells. Results of an electrophoretic mobility shift assay demonstrated that NN-B-5 interrupted the formation of alpha-trans-induction factor/C1/Oct-1/GARAT multiprotein/DNA complexes. The mechanisms of antiviral action of NN-B-5 seem to be mediated, at least in part, through inhibition of immediate early transcripts, such as ICP0 and ICP4 mRNA and then blocking of all downstream viral products accumulation and progeny HSV-1 production.

Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 confers efficient viral replication

We showed previously that infection of herpes simplex virus type 1 (HSV-1) rapidly induced the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, in the amnion cell line FL. Thus, HSV-1 suppresses the interferon (IFN) signaling pathway at the step of IFN-induced phosphorylation of janus kinases during an early infection stage. In the present study, we examined SOCS3 induction by HSV-1 infection in several types of human cell lines. FL cells and the T-cell line CCRF-CEM strongly induced SOCS3 during HSV-1 infection. The virus rapidly propagated in both cell lines and produced a lytic infection. On the other hand, the monocytic cell lines U937 and THP-1, and the B-cell line AKATA showed neither SOCS3 induction nor suppression of IFN-induced STAT1 phosphorylation during HSV-1 infection. These cell lines resulted in a persistent or prolonged infection, which continuously produced a low titer of infectious virus. The induction of SOCS3 by HSV-1 should occur via STAT3 activation immediately after HSV-1 infection. SOCS3 induction was inhibited by the addition of a Jak3 inhibitor WHI-P131. Treatment with WHI-P131 or transfection of antisense oligonucleotides specific for SOCS3 dramatically suppressed replication of HSV-1 in FL cells. The suppression of viral replication by WHI-P131 was released in the presence of neutralizing anti-IFN-α and anti-IFN-β antibodies. In conclusion, suppression of IFN signaling by HSV-1-induced SOCS3 is required for efficient replication and lytic infection of HSV-1. The SOCS3 induction varied among cell lines, indicating that it is an important factor determining the cell type specificity of efficient HSV-1 replication.

Factors That Limit the Effectiveness of Herpes Simplex Virus Type 1 for Treatment of Oral Cancer in Mice

Although the growth of experimental oral cancers can be inhibited by infection with the herpes simplex virus type 1 (HSV-1), the effect is incomplete. To define factors that might limit the effectiveness of the virus, we examined the roles of the innate immune system and the replication status of the tumor cells. AT-84 tumors were induced in strains of mice that had specific immune defects and were treated with the virus. Explanted tumors and tumor cells in culture were also infected. No differences in viral replication or in the effect of virus on the tumor were seen between mice with a lack of T or B lymphocytes, natural killer cells, phagocytic spleen cells, or complement. The virus did not replicate significantly more in tumors that were maintained as explants. Immediately after recovery of cells from a tumor the proportion of cells in the S phase was around 18%, and replication of virus in those cells was very limited. After 3 weeks in culture, the proportion in S had increased to 50% and both the recovery of virus from the cells and the toxic effect of the virus on the cells had increased significantly. The innate immune system thus seemed to have a minimal effect on replication of HSV-1 when used as an oncolytic virus for oral cancers in mice. Instead, the fraction of cells in the S phase was important. Because human oral cancers, like mouse tumors, have a low fraction of cells in the S phase, it is likely that the in vivo use of HSV-1 as cancer therapy will be limited by the replication of the virus.

Spectrum of antiviral activity of o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS)

Since some antiviral drugs have a broad spectrum of action, the aim of this study was to assess whether o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently described inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, has an effect on the replication of other retroviruses, (−) and (+) RNA viruses and DNA viruses. APHS did not affect the replication of feline immunodeficiency virus, HIV-2 and a HIV-1 strain resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI). APHS could also not inhibit the replication of the RNA viruses, respiratory syncytium virus or mouse hepatitis virus. In contrast, APHS did inhibit the replication of wild-type herpes simplex virus type 1 (HSV-1) as well as acyclovir-resistant HSV-1 and HSV-2 mutant. These results suggest that APHS is a NNRTI of HIV-1 replication, but not HIV-2 replication, and that APHS is an inhibitor of both HSV-1 and HSV-2 replication.

Mathematical analysis demonstrates that interferons- [formula omitted] and - [formula omitted] interact in a multiplicative manner to disrupt herpes simplex virus replication

Several studies suggest that the innate interferons (IFNs), IFN- α and IFN- β , can act in concert with IFN- γ to synergistically inhibit the replication of cytomegalovirus and herpes simplex virus type 1 (HSV-1). The significance of this observation is not yet agreed upon in large part because the nature and magnitude of the interaction between IFN- α / β and IFN- γ is not well defined. In the current study, we resolve this issue by demonstrating three points. First, the hyperbolic tangent function, tanh ( x), can be used to describe the individual effects of IFN- β or IFN- γ on HSV-1 replication over a 320,000-fold range of IFN concentration. Second, pharmacological methods prove that IFN- β and IFN- γ interact in a greater-than-additive manner to inhibit HSV-1 replication. Finally, the potency with which combinations of IFN- β and IFN- γ inhibit HSV-1 replication is accurately predicted by multiplying the individual inhibitory effects of each cytokine. Thus, IFN- β and IFN- γ interact in a multiplicative manner. We infer that a primary antiviral function of IFN- γ lies in its capacity to multiply the potency with which IFN- α / β restricts HSV-1 replication in vivo. This hypothesis has important ramifications for understanding how T lymphocyte-secreted cytokines such as IFN- γ can force herpesviruses into a latent state without destroying the neurons or leukocytes that continue to harbor these viral infections for the lifetime of the host.

Herpes simplex virus type 2 encodes a heat shock protein homologue with apoptosis regulatory functions

The decision to undergo apoptosis lies in the balance between pro- and anti-apoptotic proteins. Since virus replication relies on the cellular machinery, viruses have evolved various strategies to alter this balance. They target the Bcl-2 and signaling protein kinase (PK) apoptosis modulatory families by encoding homologues or altering the expression of the cellular proteins. The heat shock proteins (Hsp) are emerging as a new family of apoptosis modulatory proteins and are also a target of virus modification. Hsp function in protein folding and activation, often assisted by co-chaperones. They complex with nascent or damaged proteins and chaperone them for refolding and resumption of function, or for proteosomal degradation. Until recently, Hsp were considered strictly anti-apoptotic, possibly by virtue of their contribution to the removal of damaged and undesirable client proteins. However, recent studies have also begun to associate the Hsp with pro-apoptotic functions. Herpes simplex virus type 2 (HSV-2) encodes two proteins homologous to Hsp family members. One of these, known as ICP10PK, is a homologue to a newly cloned Hsp (H11) and modulates virus-induced apoptosis. ICP10PK is unique among the viral proteins that regulate apoptosis in that it targets all the families of apoptosis modulatory proteins. It activates the ERK signaling pathway, stabilizes Bcl-2 and upregulates Hsp70 and Hsp27 as well as the Hsp70 co-chaperone Bag-1. Its ability to commander these families of apoptosis regulators is required for HSV-2 replication and latency establishment/reactivation.

Antiviral activity of the volatile oils of Melissa officinalis L. against Herpes simplex virus type-2

Melissa officinalis L. (Lamiaceae) has been used in a variety of practical applications in medical science. Our objective in the current study was to determine the effects of the volatile oil components of M. officinalis on Herpes simplex virus type 2 (HSV-2) replication in HEp-2 cells. Four different concentrations (25, 50, 100, 150 and 200 microg/ml) of volatile oils were examined. Experiments were carried out using HEp-2 cells. M. officinalis volatile oil was found to be non-toxic to HEp-2 cells up to a concentration of 100 micro/ml. It was, however, found to be slightly toxic at a concentration over of 100 microg/ml. The antiviral activity of non-toxic concentrations against HSV-2 was tested. The replication of HSV-2 was inhibited, indicating that the M. officinalis L. extract contains an anti-HSV-2 substance.

RNA binding and R-loop formation by the herpes simplex virus type-1 single-stranded DNA-binding protein (ICP8).

In an effort to decipher the molecular mechanisms of homologous recombination during herpes simplex virus type-1 replication, we recently demonstrated that the virus-encoded single-stranded (ss) DNA-binding protein (ICP8) promotes the salt-dependent assimilation of ssDNA into a homologous plasmid, resulting in the formation of a displacement loop. In this paper, the results presented show for the first time a direct interaction between ICP8 and RNA. ICP8 binds to RNA with positive cooperativity but with approximately 5-fold lower affinity than to ssDNA. In addition, competition experiments indicate that the dissociation rate of ICP8 from RNA is faster than from ssDNA, although it is also dependent on the nature of the challenger. Importantly, ICP8 can promote the salt-dependent assimilation of RNA into a homologous acceptor plasmid to generate a joint molecule in which the RNA is stably paired with the complementary strand of the acceptor DNA, indicative of an R-loop. These findings have important implications on the role of ICP8 in mediating recombination reactions using viral transcripts. The RNA-binding activity of ICP8 also provides a molecular basis for its role in the regulation of viral gene expression.

Inhibition of human herpes simplex virus type 2 by interferon γ and tumor necrosis factor α is mediated by indoleamine 2,3-dioxygenase

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-γ and TNF-α. The antiviral effect of IFN-γ is enhanced in the presence of TNF-α, while stimulation by TNF-α alone did not induce antiviral activity. We found that IFN-γ induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-γ-induced IDO activity was enhanced in the presence of TNF-α. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-γ and the combination of IFN-γ and TNF-α. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-α are dependent on IDO activation.

Valacyclovir treatment ameliorates the persistently increased pentylenetetrazol-induced seizure susceptibility in mice with herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is an important pathogen related to epilepsy. We have shown previously that corneal inoculation of mice with HSV-1 causes acute spontaneous behavioral and electrophysiological seizures and increases hippocampal excitability and kainite-induced seizure susceptibility. In this study, we aimed to determine whether early-life HSV-1 infection in mice might cause short- and long-term enhanced susceptibility to pentylenetetrazol (PTZ)-induced seizures and to evaluate whether early antiviral drug therapy was effectively ameliorating this deficit. Seizure threshold was calculated by the latency of onset of the myoclonic jerk, generalized clonus, and maximal tonic-clonic convulsion. We demonstrate that the localization of viral antigens was predominantly within the bilateral temporal areas (amygdala, piriform, and entorhinal cortex) of HSV-1-infected mice. We also present evidence that mice of all HSV-1-infected groups had a shorter latency and higher severity to PTZ-induced seizures than in age-matched, mock-infected controls. Treatment of HSV-1-infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced a dose-dependent decrease in the signs of neurological deficits, pathological damages, and PTZ-induced seizure severity. Our results are consistent with the hypothesis that early-life HSV-1 infection leads to persistent enhancement of neuronal excitability in limbic circuits, which could result in an overall increased propensity to induce seizures later in life. Additionally, prompt optimal antiviral therapy effectively decreases seizure susceptibility in HSV-1-infected mice by limiting the level of viral replication and inflammatory response induced by virus. The present study provides not only experimental evidence, but also a new therapeutic strategy in HSV-1-associated human epilepsy.

A non-consensus branch point plays an important role in determining the stability of the 2-kb LAT intron during acute and latent infections of herpes simplex virus type-1

Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in sensory neurons of the peripheral nervous system. During HSV latency, the latency-associated transcripts (LATs) are the only viral transcripts abundantly expressed. The most abundant form of LATs is a 2-kb stable intron spliced from a primary transcript (mLAT). It has been previously reported that a non-consensus branch point influences the stability of the intron (in vitro) in cells transfected with plasmid constructs (J. Virol. 71 (1997) 5849; J. Virol. 71 (1997) 4199). However, it is unknown whether this branch point is important in determining LAT stability in vivo (in the context of virus). To study the role of this stable intron in HSV-1 infection, we have constructed a mutant virus KOS-CONS in which the branch point has been mutated to consensus branch point nucleotides. The accumulation of the 2-kb intron in KOS-CONS-infected cells was greatly reduced. The LAT intron was not detectable in KOS-CONS-infected mouse trigeminal ganglia (TG) during acute and latent phase infection by Northern blot analysis. Replication of the KOS-CONS and the wild-type KOS viruses on Vero cells was determined to be similar, as was the level of HSV-1 DNA in mouse trigeminal ganglia during acute and latent phase infection. Using the mouse TG explant model, the reactivation pattern of both viruses was shown to be similar. Our data suggest that the unique branch point plays a significant role in determining the stability of LAT intron in vivo, but that the stability of the intron does not appear to affect HSV-1 replication, the establishment of latency, or viral reactivation.

The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice.

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.

The UL12.5 gene product of herpes simplex virus type 1 exhibits nuclease and strand exchange activities but does not localize to the nucleus.

The herpes simplex virus type 1 (HSV-1) alkaline nuclease, encoded by the UL12 gene, plays an important role in HSV-1 replication, as a null mutant of UL12 displays a severe growth defect. Although the precise in vivo role of UL12 has not yet been determined, several in vitro activities have been identified for the protein, including endo- and exonuclease activities, interaction with the HSV-1 single-stranded DNA binding protein ICP8, and an ability to promote strand exchange in conjunction with ICP8. In this study, we examined a naturally occurring N-terminally truncated version of UL12 called UL12.5. Previous studies showing that UL12.5 exhibits nuclease activity but is unable to complement a UL12 null virus posed a dilemma and suggested that UL12.5 may lack a critical activity possessed by the full-length protein, UL12. We constructed a recombinant baculovirus capable of expressing UL12.5 and purified soluble UL12.5 from infected insect cells. The purified UL12.5 exhibited both endo- and exonuclease activities but was less active than UL12. Like UL12, UL12.5 could mediate strand exchange with ICP8 and could also be coimmunoprecipitated with ICP8. The primary difference between the two proteins was in their intracellular localization, with UL12 localizing to the nucleus and UL12.5 remaining in the cytoplasm. We mapped a nuclear localization signal to the N terminus of UL12, the domain absent from UL12.5. In addition, when UL12.5 was overexpressed so that some of the enzyme leaked into the nucleus, it was able to partially complement the UL12 null mutant.

Recruitment of cellular recombination and repair proteins to sites of herpes simplex virus type 1 DNA replication is dependent on the composition of viral proteins within prereplicative sites and correlates with the induction of the DNA damage response.

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.

Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9

AbstractNatural interferon-producing cells (IPCs) specialize in the production of high levels of type 1 interferons (IFNs) in response to encapsulated DNA and RNA viruses. Here we demonstrate that the secretion of type 1 IFN in response to herpes simplex virus type 1 (HSV-1) in vitro is mediated by the toll-like receptor 9 (TLR9)/MyD88 pathway. Moreover, IPCs produce interleukin-12 (IL-12) in response to HSV-1 in vitro, which is also dependent on TLR9/ MyD88 signaling. Remarkably, though TLR9/MyD88-deficiency abrogates IPC responses to HSV-1 in vitro, mice lacking either MyD88 or TLR9 are capable of controlling HSV-1 replication in vivo after local infection, demonstrating that TLR9- and MyD88-independent pathways in cells other than IPCs can effectively compensate for defective IPC responses to HSV-1.

Metabolic and Pharmacological Characteristics of the Bicyclic Nucleoside Analogues (BCNAs) as Highly Selective Inhibitors of Varicella—Zoster Virus (VZV)

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication.

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.