An investigation of herpes simplex virus type 1 latency in a novel mouse dorsal root ganglion model suggests a role for ICP34.5 in reactivation.

After a primary lytic infection at the epithelia, herpes simplex virus type 1 (HSV-1) enters the innervating sensory neurons and translocates to the nucleus, where it establishes a quiescent latent infection. Periodically, the virus can reactivate and the progeny viruses spread back to the epithelium. Here, we introduce an embryonic mouse dorsal root ganglion (DRG) culture system, which can be used to study the mechanisms that control the establishment, maintenance and reactivation from latency. Use of acyclovir is not necessary in our model. We examined different phases of the HSV-1 life cycle in DRG neurons, and showed that WT HSV-1 could establish both lytic and latent form of infection in the cells. After reactivating stimulus, the WT viruses showed all markers of true reactivation. In addition, we showed that deletion of the γ(1)34.5 gene rendered the virus incapable of reactivation, even though the virus was clearly able to replicate and persist in a quiescent form in the DRG neurons.

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells.

Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral gamma 34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 gamma 34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a gamma 34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the gamma 34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers.

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals.

We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

HFE downregulates iron uptake from transferrin and induces iron-regulatory protein activity in stably transfected cells.

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular "labile iron pool." The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

A herpesvirus ribosome-associated, RNA-binding protein confers a growth advantage upon mutants deficient in a GADD34-related function.

The herpes simplex virus type 1 gamma34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (gamma34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation.

Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene.

Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.

A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function.

Novel suppressor variants of conditionally lethal HSV-1 gamma34.5 deletion mutants have been isolated which exhibit restored ability to grow on neoplastic neuronal cells. Deletion of the viral gamma34.5 genes, whose products share functional similarity with the cellular GADD34 gene, renders the virus non-neurovirulent and imposes a block to viral replication in neuronal cells. Protein synthesis ceases at late times post-infection and the translation initiation factor eIF2alpha is phosphorylated by the cellular PKR kinase [Chou et al. (1990) Science, 252, 1262-1266; (1995) Proc. Natl Acad. Sci. USA, 92, 10516-10520]. The suppressor mutants have overcome the translational block imposed by PKR. Multiple, independent isolates all contain rearrangements within a 595 bp element in the HSV-1 genome where the unique short component joins the terminal repeats. This alteration, which affects the production of the viral mRNA and protein from the Us11 and Us12 genes, is both necessary and sufficient to confer the suppressor phenotype on gamma34.5 mutant viruses. HSV-1 thus encodes a specific element which inhibits ongoing protein synthesis in the absence of the viral GADD34-like function. Since this inhibition involves the accumulation of phosphorylated eIF2alpha, the element identified by the suppressor mutations may be a discrete PKR activator. Activation of the PKR kinase thus does not proceed through a general, cellular 'antiviral' sensing mechanism. Instead, the virus deliberately activates PKR and encodes a separate function which selectively prevents the phosphorylation of at least one PKR target, eIF2alpha. The nature of this potential activator element, and how analogous cellular elements could affect PKR pathways which affect growth arrest and differentiation are discussed.

The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.

The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.

For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, E1 and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer of both transcription and replication and is known to help E1 bind cooperatively to the origin of DNA replication. E1 is sufficient for replication in extracts prepared from permissive cells, but the activity is enhanced by E2. Here we show that purified E1 can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of E1 to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, E1-dependent viral DNA replication is origin-dependent, but at elevated E1 concentrations, replication can occur on non-origin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication.

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.

Statistical analysis of the incidence of positives in the examination of parasitological specimens.

Results of the examination of 2,000 parasitological specimens were analyzed for differences between results obtained by a technologist working in parasitology only and those obtained by general microbiologists who were also performing other laboratory work. The relative share of specimens determined to be positive by the specialist was approximately twice as high as the relative share determined by the general microbiologists.

A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.

We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.

Activation of BPV-1 replication in vitro by the transcription factor E2.

Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator.

The mechanism by which transcription factors stimulate DNA replication in eukaryotes is unknown. Bovine papillomavirus DNA synthesis requires the products of the viral E1 gene and the transcriptional activator protein encoded by the E2 gene. Experimental data showed that the 68-kilodalton (kD) E1 protein formed a complex with the 48-kD E2 transcription factor. This complex bound specifically to the viral origin of replication, which contains multiple binding sites for E2. Repressor proteins encoded by the E2 open reading frame failed to complex with E1 suggesting that the 162-amino acid region of E2 that participates in transactivation contained critical determinants for interaction with E1. The physical association between a replication protein and a transcription factor suggests that transcriptional activator proteins may function in targeting replication initiator proteins to their respective origins of replication.

Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication.

Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.

Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication.

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.

Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen.

A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed.

Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

The origin-specific DNA-binding domain of simian virus 40 large T antigen was analyzed, and its C-terminal boundary was found to be at or before amino acid 259. This does not include the zinc finger structural motif located at amino acids 302 to 320 (J. M. Berg, Science 232:485-486, 1986). Interestingly, N-terminal fragments of 266 and 272 amino acids and larger displayed dramatically reduced origin-binding activity. In addition, the specific DNA-binding properties of truncated proteins purified from both bacterial and mammalian sources were compared. Truncated T antigens from mammalian cells bound specific DNA fragments more efficiently than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulating the DNA-binding activity of large T antigen.