Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

Self Cite

Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.A deno-associated virus type 2 (AAV2) is a small, nonenveloped parvovirus with a single-stranded DNA genome of 4.7 kb (52). In the absence of a helper virus, AAV2 establishes a latent infection characterized by site-specific integration of the viral genome into the AAVS1 site on human chromosome 19 (72). In the presence of a helper virus, AAV2 can replicate productively in the host cell nucleus. AAV2 DNA replication occurs at discrete sites in the nucleus, termed replication compartments (RCs). During the course of infection, several small RCs rapidly expand and fuse to large structures, which displace the cellular chromatin and fill the entire cell nucleus (28,35,37,79,91). AAV2 RCs contain AAV2 proteins, as well as defined helper virus proteins and cellular proteins (3,35,63,65,75,79,90,91). Replicating AAV2 has inhibitory effects on both the host cell (9,41,68,71,73,74,100,101) and the helper virus (5,30,31,34,40,44,61,84,100).One of the helper viruses for AAV2 replication is herpes simplex virus 1 (HSV-1) (14). The minimal HSV-1 helper factors for AAV2 replication from plasmid substrates include the helicaseprimase complex encoded by UL5, UL8, and UL52 and the major DNA binding protein ICP8 (3) (90). Besides viral helper factors, the fate of AAV2 replication also depends on cellular proteins. Recently, cellular proteins have been identified that interact with AAV2 Rep78/68 in adenovirus (Ad)-or HSV-1-supported AAV2 replication (63,65). Of these, the largest functional categories correspond to cellular proteins which are involved in DNA metabolism, including DNA replication, repair, and chromatin modification.There is accumulating evidence that the DNA damage response (DDR) pathways play central roles in viral replication (92). Control of DDR signaling may be a mechanism to prevent apoptosis and/or stop cell cycle progression (92)...

Section: Discussionmentioning

confidence: 58%

mentioning

confidence: 99%

Adeno-Associated Virus Type 2 Modulates the Host DNA Damage Response Induced by Herpes Simplex Virus 1 during Coinfection

Vogel

Seyffert

Strasser

et al. 2012

Self Cite

“…This approach may be used to address several open questions in HSV-1 biology, for example, the spatial organization of capsid assembly and maturation by fluorescent labeling of several components of the pro-capsid and those of mature capsids. In addition, fluorescently labeled virus proteins may be combined with systems for the live visualization of viral DNA, as previously described for several viruses, such as HSV-1, Epstein-Barr virus, and adeno-associated virus (1,23,28,29,58), to specifically assess the dynamics of the association of viral proteins with viral DNA. Finally, the simultaneous fluorescent labeling of capsid, tegument, and envelope components may prove useful for the study of virus trafficking, for example, to assess and compare the composition of virions transported in an anterograde versus retrograde direction within axons (2-4, 20, 40).…”

Section: Discussionmentioning

confidence: 99%

“…At the indicated time points, the cells were washed once with cold PBS and fixed with 3.7% formaldehyde in PBS for 15 min at RT, and the fixation was stopped with 0.1 M glycine in PBS for 5 min at RT. Immunofluorescence staining, DAPI staining, and embedding of cells were performed as described previously (29) except that the cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min at RT and that 0.2 mg/ml human IgG (Sigma-Aldrich, Buchs, Switzerland) was included in the blocking solution when cells were stained with antibodies of rabbit origin. Primary antibodies were used at the following dilutions: anti-HSV-1 ICP8 MAb 7381 (kindly provided by R. D. Everett, MRC Virology Unit, Glasgow, United Kingdom), 1:500 (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Live Visualization of Herpes Simplex Virus Type 1 Compartment Dynamics

Oliveira

Glauser

Laimbacher

et al. 2008

Self Cite

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.The herpes simplex virus type 1 (HSV-1) virion consists of three different compartments, capsid, tegument, and envelope. The icosahedral capsid has a diameter of 125 nm and contains the virus genome, a double-stranded DNA of 152 kbp. The structural basis of the capsid are the 162 capsomers, which include 150 hexons and 12 pentons (47). The capsomers are connected in groups of three by a complex formed with two copies of VP23 and one copy of VP19c (47,54,68). The hexons are composed of six molecules of the major capsid protein VP5. Eleven of the 12 pentons are composed of five molecules of VP5, while 1 of the 12, the so-called portal, is a cylindrical structure of 12 molecules of UL6 (46). Also involved in capsid assembly, but not physical components of the capsids, are the scaffold polypeptides VP22a, VP21, and the serine protease, VP24, which is required for capsid maturation (9,26,38,51). Six copies of VP26, a 12-kDa polypeptide encoded by the UL35 gene, occupy the tips of each hexon and thus decorate the surface of the capsid (42, 69). Although not essential...

“…In addition, the HSV-1 IE proteins ICP4 and ICP0, the E protein complex forming the HSV-1 polymerase (UL30 and UL42), and the US1 gene product strongly enhance AAV2 replication (26). AAV2 has developed strategies to inhibit helper virus replication, likely to reduce competition (24,(28)(29)(30)(31)(32)(33). For example, expression of the AAV2 nonstructural proteins Rep68 and Rep78 alone leads to significant inhibition of 28).…”

mentioning

confidence: 99%

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

Seyffert

Glauser

Tobler

et al. 2015

Self Cite

e Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase-and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Repbinding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.A deno-associated virus type 2 (AAV2) is a nonpathogenic human parvovirus with a unique biphasic life cycle. In the absence of a helper virus, AAV2 establishes a latent infection, while in the presence of a helper virus, such as adenovirus type 2 (AdV2), herpes simplex virus 1 (HSV-1), or human papillomavirus 16 (HPV-16), it undergoes lytic replication (1-4). The AAV2 genome is a single-stranded DNA (ssDNA) molecule of 4,680 nucleotides, which is packaged into an icosahedral capsid with a diameter of approximately 20 nm (5). The genome harbors two clusters of genes, rep and cap, which are flanked by inverted terminal repeats (ITRs). The ITRs form hairpin structures and contain a Rep-binding site (RBS) and a terminal resolution site (trs), which together act as the viral origin of DNA replication (6, 7). The cap gene is transcribed from the p40 promoter and encodes the capsid proteins VP1, VP2, and VP3, which differ in their N termini due to alternative start codons (8, 9). In addition, a nested open reading frame (ORF) within the cap gene encodes a protein designated assembly-activating protein (AAP), which is believed to be required for AAV2 capsid assembly in the nucleolus (10, 11). The rep gene encodes the Rep proteins, which are synthesized in four different forms due to transcription from two different promoters, p5 and p19, and alternative splicing of an intron near the C-terminal end (12). The different Rep proteins are termed Rep40, Rep52, Rep68, and Rep78 according to their apparent molecular weights. The Rep proteins are involved in diverse processes during the viral life cycle, such as DNA replication, regulation of gene expression, genome packaging, and site-specific genomic integration (13-18).HSV-1 belongs to the subfamily of the Alphaherpesvirinae and is the reagent causing mucosal eruptions at the site of infection, which can reoccur at the same site upon reactivation from latency (19,20). The HSV-1 virion is built up by three structural components, the capsid, the tegument, and the surrounding envelope. The viral genome is a linear double-stranded DNA (dsDNA) molecule 152 kb in size and has a unique structure. It is divided into two covalently joined segments, which contain unique segments (unique long [U L ] and unique short [U S ]) and inverted repeat regions (TR L , IR L , IR S , and TR S ). The IR sequences link the L and S segments (Fig. 1A). HSV-1 gene expression and replication occur in a temporally regulated cascade: immediate early (IE), early (E), and late. IE proteins exhibit mainly regulatory...