A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.     

A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication.

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.     

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.     

一种包装AAV2/5的重组单纯疱疹病毒的构建

为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。     

Role of the herpes simplex virus helicase-primase complex during adeno-associated virus DNA replication

A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.     

新型重组AAV5/5载体及包装系统

本研究组建了一种可用于规模化生产的以重组单纯疱疹病毒为辅助病毒的AAV5/5载体包装系统。首先,将5型腺相关病毒 (AAV5) 的rep和cap基因插入I型单纯疱疹病毒 (HSV-1) 基因组非必需基因UL2中,获得重组病毒rHSV1-rep5cap5。其次,构建一种携带AAV5 ITR的通用型载体质粒pAAV5neo,将报告基因EGFP插入pAAV5neo中,得到pAAV5neo-EGFP质粒。将pAAV5neo-EGFP质粒导入BHK-21细胞,用G418选择培养,挑选出表达EGFP并在重组病毒rHSV1-rep5cap5感染下能高效产生rAAV5/5-EGFP的单克隆载体细胞株C020。用rHSV1-rep5cap5感染C020细胞制备rAAV5/5-EGFP,用“氯仿处理-聚乙二醇/氯化钠-氯仿抽提”方法粗纯化rAAV5/5-EGFP。用100 kDa分子量截流超滤方法进一步纯化和浓缩,获得高纯度的rAAV5-EGFP。SDS-PAGE电泳分析可见3条特征性外壳蛋白带。电镜分析显示病毒颗粒以实心颗粒为主。用rAAV5/5-EGFP病毒按1×105 vg/cell感染体外培养的HEK293细胞,可见30％细胞呈现绿色荧光。本研究提出了一种高效AAV5/5载体生产系统和纯化方法,为重组AAV5载体的进一步应用提供了基础。     

Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication

In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.     

DNA-binding activity of adeno-associated virus Rep is required for inverted terminal repeat-dependent complex formation with herpes simplex virus ICP8

Herpes simplex virus (HSV) helper functions for (AAV) replication comprise HSV ICP8 and helicase-primase UL5/UL52/UL8. Here we show that N-terminal amino acids of AAV Rep78 that contact the Rep-binding site within the AAV inverted terminal repeat (ITR) are required for ternary-complex formation with infected-cell protein 8 (ICP8) on AAV single-strand DNA (ssDNA) in vitro and for colocalization in nuclear replication domains in vivo. Our data suggest that HSV-dependent AAV replication is initiated by Rep contacting the AAV ITR and by cooperative binding of ICP8 on AAV ssDNA.     

High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.     

Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep(-) vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep(+), but not the rep(-), hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.     

Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression.

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.     

Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication.

Adeno-associated virus (AAV) contains a multifunctional nonstructural gene, rep, which is required for AAV DNA replication and has pleiotropic effects on positive and negative regulation of gene expression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conserved region is the consensus sequence for a purine nucleotide binding site. We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced the mutant Rep proteins, indicating that these proteins are autoregulated. Furthermore, the mutant gene was unable to replicate but was able to inhibit in trans wild-type AAV DNA replication. Thus, pNTC23 represents a dominant negative mutant of AAV. These results suggest that rep has separate functional domains important for DNA replication.     

Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected primate cells

Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, gamma-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage.