SV40 Virus Co-Opts the Host DNA Damage Response
By: Carol A. Rouzer, VICB Communications
Published: April 30, 2013
Activation of the host kinases ATM and ATR during SV40 infection facilitates the synthesis of functional, unit-length viral genomes.
The DNA damage response (DDR) is a critical process that promotes genomic stability and insures that damaged cells do not progress through the cell cycle. However, this cellular machinery can be co-opted by viruses to facilitate replication of their DNA. Now, VICB member Ellen Fanning and her laboratory report the results of their studies using small molecule inhibitors to explore the role of two DDR enzymes, ATM (ataxia telangiectasia-mutated) and ATR (ATM- and Rad3-related), in the replication of simian virus 40 (SV40) DNA. [G. A. Sowd, et al. (2013) PLoS Pathogens, 9, e1003283].
SV40 is a monkey virus closely related to viruses prevalent in human populations. Studies of SV40 infection in monkey kidney cells reveals that viral genome replication begins at the origin of replication where binding of the SV40 T antigen (Tag) protein, a helicase, leads to unwinding of the DNA. Host cell proteins, including replication protein A (RPA), primase-polymerase α (primase-Pol α) and replication factor C (RFC) synthesize an RNA primer and begin the process of replicating the leading strand (Figure 1A). Replacement of primase-Pol α with Pol δ facilitates further extension of the strand. At the same time, lagging-strand synthesis begins, occurring in short Okazaki fragments, which are later ligated together. The SV40 genome is circular, and replication occurs on both strands of the DNA simultaneously in a process known as theta replication, since it leads to formation of intermediates that resemble the Greek letter θ (Figure 1B).
Figure 1. Replication of the SV40 genome. (A) The process begins at the SV40 origin of replication with the binding of the viral Tag protein, a helicase that unwinds the DNA. Host proteins RPA, primase-Pol α and RFC work together to synthesize a primer and start leading strand DNA synthesis. Replacement of primase-Pol α with Pol δ promotes extension of the strand. Then, primer synthesis followed by extension replicates the lagging strand in short Okazaki fragments, which are ligated together. Note that synthesis occurs in both directions on both strands of the virus. Image reproduced from Wikimedia Commons. It is a work of the National Institutes of Health and is in the public domain. Source: Molecular Cell Biology, 4th edition, Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000. (B) The process shown in (A) occurs on the circular viral DNA producing an intermediate that resembles the Greek letter θ. Red and orange represent the parental DNA strands, and the black bar designates the origin of replication. Blue and green indicate the newly synthesized strands.
ATM and ATR are both protein kinases that phosphorylate consensus SQ/TQ motifs in target proteins, including the histone H2AX. Phosphorylated H2AX (γH2AX) helps to recruit DNA repair proteins and promotes the activation of Chk1 and Chk2, leading to cell cycle arrest. Activation of ATM is triggered by DNA double-strand breaks (DSBs), oxidative damage, and alterations in chromatin structure, while RPA-bound single-stranded DNA activates ATR. Prior studies had shown that SV40 can replicate in the absence of functional ATM or ATR, but the result is a reduction in unit-length viral DNA. The Fanning lab’s goal was to understand how this occurs, and to determine the role of these two DDR proteins in SV40 replication. First, they infected monkey kidney BSC40 cells with SV40 virus and used immunofluorescence microscopy to observe the cellular localization of replication-related proteins (Figure 2). They found discrete regions in the nuclei of infected cells that labeled brightly with antibodies against viral Tag. These regions were also labeled by probes directed against ethynyl-2′-deoxyuridine (EdU), which had been incorporated into newly synthesized DNA. The host proteins Pol δ, RFC-1, and proliferating cell nuclear antigen (PCNA), all known to be involved in SV40 replication, co-localized with Tag and EdU. In contrast, Tag did not co-localize with Cdc45, which plays a role in host cell, but not viral DNA replication. These initial results indicated that labeling of Tag could be used to identify sites of SV40 DNA replication within the cells.
Figure 2. Fluorescence microscopy of the nuclei of SV40-infected BSC40 cells. Cells were stained for the viral Tag (green), host cell replication proteins or newly synthesized DNA as indicated (red), and total DNA (blue). Merged staining shows co-localization of Tag with host proteins PCNA, Pol δ, and RFC-1 and with newly synthesized DNA labeled with EdU. Image reproduced from G. A. Sowd, et al. (2013) PLoS Pathogens, 9, e1003283, under the creative commons license.
To determine whether SV40 replication alone could activate the DDR, the investigators used the pMini SV40 plasmid to deliver the viral genome to the cells in the absence of viral infection. Indeed, transfection of BSC40 cells with the plasmid carrying the wild-type SV40 genome induced the formation of γH2AX at the site of SV40 replication. Immunoblot analysis revealed phosphorylated Chk1 and Chk2 in pMini SV40-transfected cells, providing further confirmation of DDR activation. In contrast, transfection of the cells with plasmids bearing a mutation in the Tag gene that inactivates helicase activity, or a mutation that inactivates the origin of replication led to Tag expression in the cells but no SV40 DNA replication and no activation of the DDR (Figure 3).
Figure 3. Fluorescence microscopy of the nuclei of BSC40 cells transfected with pMini SV40 carrying the wild-type SV40 genome (wt), an SV40 genome coding for the D474N mutation in Tag, which inactivates the helicase activity (D474N), or a mutation that inactivates the origin of replication (In-1). Mock transfected cells served as negative controls. All plasmids could trigger Tag expression, but only the wild-type plasmid was competent for SV40 DNA replication. Only the wt plasmid activated the DDR, as indicated by γH2AX staining that co-localized with Tag. Image reproduced from G. A. Sowd, et al. (2013) PLoS Pathogens, 9, e1003283, under the creative commons license.
Having established that SV40 genome replication alone is sufficient to activate the DDR, the Fanning lab used Ku-55933, a selective inhibitor of ATM, to investigate the precise role of this kinase. They found that addition of Ku-55933 to cells during the early stages of viral infection resulted in a reduction in viral replication centers visible by fluorescence microscopy. However, when the inhibitor was present during the later stages or throughout the infection, formation of normal replication centers was virtually eliminated (Figure 4). These results suggest that, while ATM activity facilitates SV40 genome replication during the early stages of infection, it is absolutely required during the later stages. Further studies showed that early exposure of SV40-infected cells to Ku-55933 led to reduction in the total amount of SV40 DNA synthesized, but that most of the DNA was in the form of unit-length genomes. In contrast, the presence of the compound late in the infection led to a marked increase in the formation of large concatemeric DNA structures. These results suggested that, late in infection, ATM activity is needed to produce unit-length viral genomes.
Figure 4. Fluorescence microscopy of nuclei of infected cells grown in the presence of Ku-55933, which was added early, late, or during the full coarse of the infection. Note that addition late or throughout the infection leads to loss of normal discrete foci of viral replication. Image reproduced from G. A. Sowd, et al. (2013) PLoS Pathogens, 9, e1003283, under the creative commons license.
To further understand the role of ATM in SV40 replication, the Fanning lab used two-dimensional agarose gel electrophoresis to examine the structure of replication intermediates formed in the presence and absence of Ku-55933. Theta replication produces structures known as Cairns intermediates (Figure 1B and 5). Cutting these intermediates using restriction enzymes can produce either a double Y structure or a bubble structure, depending on the location of the cut. Accordingly, BgII, which cleaves at the origin of replication, produced double Y structures from SV40 replication intermediates produced by infected cells in the absence of Ku-55933, while BamHI, which cleaves at the opposite side of the circle, produced bubble structures. However, when the same enzymes were used to cleave intermediates formed by infected cells in the presence of Ku-55933, a substantial quantity of simple Y structures and X structures were obtained in addition to more complex D-loop structures. These results suggested that, in the presence of Ku-55933, the SV40 genome was undergoing rolling circle replication, as opposed to its usual theta replication (Figure 5). Rolling circle replication proceeds around the circular viral template in one direction and can easily lead to the concatemeric structures produced in Ku-55933-treated SV40-infected cells.
Figure 5. (A) Cleavage of Cairns intermediates produced during SV40 theta replication produces double Y or bubble structures, depending on the site of cleavage. (B) A break in the Cairns intermediate at one replication fork can lead to a switch to rolling circle replication, with or without a D-loop structure. Cleavage of these intermediates can yield simple Y or more complex D-loop structures.
Similar studies using an inhibitor of ATR (ATRi) also revealed a decrease in total viral DNA production and an increase in concatemeric forms. As in the case of ATM inhibition, two-dimensional gel electrophoresis of intermediates produced by infected cells in the presence of ATRi exhibited an increase in simple Y structures. However, unlike results obtained in the presence of Ku-55933, simple Y structures produced by ATRi-treated cells showed an uneven size distribution depending on the restriction enzyme used for cleavage. This result, which was not consistent with rolling circle replication, led the investigators to conclude that although both ATM and ATR are required for synthesis of unit-length SV40 genomes, their roles are distinct, and the aberrant products formed in their absence are also distinct.
Figure 6. Proposed roles of ATM and ATR in SV40 replication. The circular DNA is shown is coiled form, as would be found in nature. Steps I through IV show the process of theta replication. A DSB at the site of one replication fork in the absence of ATM leads to unidirectional rolling circle replication. Stalling of a replication fork, followed by a break occurring when the second fork approaches the stalled fork, yields broken intermediates that are repaired by ATR. Image reproduced from G. A. Sowd, et al. (2013) PLoS Pathogens, 9, e1003283, under the creative commons license.
These findings led the Fanning group to propose that the rapid rate of DNA replication during viral infection can easily lead to strand breaks. A one-ended replication-associated DSB occurring at a replication fork could lead to loss of replication at that fork and a switch to rolling circle replication and concatemer formation at the remaining fork. Repair of this type of DSB by ATM would restore normal theta replication, leading to unit-length viral DNA. Alternatively, stalling of replication at one fork would halt DNA synthesis at that fork, but replication could continue at the other fork until it reached the stalled fork. At this point point a DSB occurs at the actively replicating fork. ATR facilitates repair of breaks occurring under these circumstances. In its absence, the result would be the unusual simple Y pattern observed on two-dimensional analysis of intermediates produced in the presence of ATRi.
Together the results confirm that SV40 genome replication leads to activation of at least some arms of the DDR, including ATM and ATR. The virus has evolved to take advantage of this host response to facilitate efficient production of functional unit-length viral genomes, even though they are produced under conditions of considerable cellular stress.