Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to enhance its infection in host cells. An essential aspect of the MVM-induced DDR is the establishment of a potent G2/M arrest. Although p53 remains activated, p21 is degraded and ATR/Chk1 signaling is disabled. We find that a surprising p21- and Chk1-independent cell cycle block is established via a novel mechanism that results in the significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function thus preventing mitotic entry. The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They are the only known viruses of vertebrates that contain single-stranded linear DNA genomes, and they continually present novel replicative DNA structures to cells during infection. This application proposes to further examine how MVM exploits the cellular DDR to prepare the nuclear environment for effective parvovirus takeover. The following aims are based on recently published findings:
Aim I. How, and for what purpose, does the MVM-induced DDR cause re-localization of the CRL4Cdt2 E3 ubiquitin ligase to APAR bodies? Efficient MVM replication requires the CRL4Cdt2 E3 ubiquitin ligase-targeted depletion of p21 to prevent its inhibitory interaction with PCNA, an essential co-factor for DNA polymerase ?, which replicates the MVM genome. This ligase also has other important targets involved in DNA replication. We propose to further characterize the status and localization of the components of the ligase complex within viral replication centers; characterize the mechanism by which p21 is depleted, Cdt1 is spared, and how this facilitates infection; and investigate potential additional roles for the ligase during infection.
Aim II. How, and for what purpose, is ATR signaling disabled during the MVM-induced DDR? Neither Chk1, a downstream target of ATR and normally an important mediator of cell cycle arrest, nor ATR itself, is activated during MVM infection, even though viral genomes bearing bound RPA - normally a potent trigger of ATR activation - accumulate in viral replication centers. It is now clear that ATR signaling is actively disabled following establishment of the MVM-induced DDR. We propose to investigate the means by which ATR is disabled, and the purpose its disabling serves during infection.
Aim III. How, and for what purpose, does the MVM-induced DDR cause depletion of cyclin B1? The MVM-induced p21- and Chk1-independent cell cycle arrest proceeds via a process unlike that seen in response to other DNA-damaging agents or other virus infections: it results in a dramatic depletion of cyclin B1 and its encoding RNA which precludes mitotic entry. We propose to determine the mechanism by which cyclin B1 RNA is depleted in infected cells, whether there is a role for the viral nonstructural proteins within the context of an ongoing DDR, and to determine the purpose this depletion serves during infection.

Public Health Relevance

The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They possess small linear single-stranded DNA (ssDNA) genomes and their replication relies extensively on host nuclear factors. Upon infection, parvoviruses induce a cellular DNA damage response (DDR) that they exploit to their replicative advantage. Our studies will clarify critical aspects o parvovirus-cell interaction, develop new paradigms in the DDR field, and deliver significant insight into fundamental aspects of viral pathogenesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Natarajan, Ramya
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University of Missouri-Columbia
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Majumder, Kinjal; Wang, Juexin; Boftsi, Maria et al. (2018) Parvovirus minute virus of mice interacts with sites of cellular DNA damage to establish and amplify its lytic infection. Elife 7:
Fuller, Matthew S; Majumder, Kinjal; Pintel, David J (2017) Minute Virus of Mice Inhibits Transcription of the Cyclin B1 Gene during Infection. J Virol 91:
Majumder, Kinjal; Etingov, Igor; Pintel, David J (2017) Protoparvovirus Interactions with the Cellular DNA Damage Response. Viruses 9: