Significance TIM-family proteins have been recently shown to promote viral entry into host cells. Unexpectedly, we discovered that human TIM-1, along with TIM-3 and TIM-4, potently inhibits HIV-1 release. We showed that TIM-1 is incorporated into HIV-1 virions and retains HIV-1 particles on the plasma membrane via phosphatidylserine (PS), a phospholipid that is exposed on the cellular plasma membrane and the viral envelope. Expression of TIM-1 inhibits HIV-1 replication in CD4 + T cells, and knockdown of TIM-3 in monocyte-derived macrophages enhances HIV-1 production. We extended this function of TIMs to other PS receptors, and demonstrated that they also inhibited release of additional viruses, including murine leukemia virus and Ebola virus. The novel role of TIMs in blocking viral release provides new insights into viral replication and AIDS pathogenesis.

We have developed a generally adaptable, novel high-throughput Viral Chromosome Conformation Capture assay (V3C-seq) for use in trans that allows genome-wide identification of the direct interactions of a lytic virus genome with distinct regions of the cellular chromosome. Upon infection, we found that the parvovirus Minute Virus of Mice (MVM) genome initially associated with sites of cellular DNA damage that in mock-infected cells also exhibited DNA damage as cells progressed through S-phase. As infection proceeded, new DNA damage sites were induced, and virus subsequently also associated with these. Sites of association identified biochemically were confirmed microscopically and MVM could be targeted specifically to artificially induced sites of DNA damage. Thus, MVM established replication at cellular DNA damage sites, which provide replication and expression machinery, and as cellular DNA damage accrued, virus spread additionally to newly damaged sites to amplify infection. MVM-associated sites overlap significantly with previously identified topologically-associated domains (TADs).

The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5’ end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVMinduced DDR is the establishment of a potent premitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others reported previously, depends upon a significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function, thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected by MVM infection, the production of nascent cyclin B1 RNA was substantially diminished at late times postinfection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein, and RNA levels normally increased in response to DNA-damaging reagents. We demonstrated that at times of reduced cyclin B1 expression during infection, there was a significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 gene promoter. Additionally, while total FoxM1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via clustered regularly interspaced short palindromic repeats (CRISPR)-enzymatically inactive Cas9 in MVM-infected cells increased both cyclin B1 protein and RNA levels, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection.IMPORTANCE Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus exploits to prepare the nuclear environment for effective takeover. An essential aspect of the MVM-induced DDR is establishment of a potent premitotic block. This block depends upon a significant, specific depletion of cyclin B1 and its encoding RNA that precludes cyclin B1/CDK1 complex functions necessary for mitotic entry. We show that reduced cyclin B1 expression is controlled primarily at the level of transcription initiation. Additionally, the essential mitotic transcription factor FoxM1 and RNA polymerase II were found to occupy the cyclin B1 gene promoter at reduced levels during infection. Recruiting a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via CRISPR-catalytically inactive Cas9 (dCas9) in MVM-infected cells increased expression of both cyclin B1 protein and RNA, implicating FoxM1 as a critical target mediating MVM-induced cyclin B1 inhibition.

29We have developed a generally adaptable, novel high-throughput Viral Chromosome

The DNA damage response (DDR) is a critical cellular network that affords cells the ability to repair DNA damage they have incurred from endogenous and exogenous sources. Recently, it has become appreciated that viruses, both DNA and RNA, can induce the DDR and have evolved the ability to interact with this ancient antiviral mechanism. Viruses can choose to inactivate or utilize this host response, which typically requires specific modulation for either case. Importantly, it has been shown that MVM utilizes this response to facilitate its replication in an ATM-dependent manner. MVM induces a DDR-dependent pre-mitotic, G2/M cell cycle block via activation of the checkpoint kinase, Chk2, and depletion of the RNA and protein of the key mitotic cyclin, cyclin B1. Unexpectedly, this cell cycle block was shown to be p21- and Chk1-independent. MVM infection results in the recruitment and activation of numerous DDR proteins including Chk2, RPA32 and p53. Upon activation, via phosphorylation, p53, a critical tumor suppressor, is known to transactivate several hundred genes including the well-characterized CDK inhibitor, p21. However, previous work demonstrated a sustained, proteasome-dependent loss of p21 during infection, which was required for efficient replication. This depletion of p21 during infection was unexpected, given the activation of p53 during infection, and because p21 is a known, potent cell cycle inhibitor. We investigated the loss of p21 during infection and found that siRNA knockdown of specific components of the CRL4Cdt2 E3 ubiquitin ligaseCul4A, DDB1 and Cdt2stabilized p21 during MVM infection. Importantly, siRNA knockdown of specific components of CRL4Cdt2 reduced viral replication. DDB1 and Cdt2, the adapter protein and substrate recognition factor of the CRL4Cdt2, respectively, were recruited to viral replication factories, termed autonomous parvovirus-associated replication (APAR) bodies, suggesting that MVM may be hijacking this important E3 ubiquitin ligase. The recruitment and utilization of this ligase is likely specific, as the APC/CCDC20 E3 ubiquitin ligase, which also targets p21 for proteasome-dependent degradation, was not recruited to APAR bodies nor did siRNA knockdown of specific components of this ligase stabilize p21 during infection. Taken together, these results suggest that MVM specifically utilizes the CRL4Cdt2 E3 ubiquitin ligase to target p21 for degradation and that the activity of this E3 ubiquitin ligase is required for efficient MVM replication. The requirements for the activity of CRL4Cdt2 gave us a hint as to why MVM would target p21 for degradation. It has been shown that CRL4Cdt2 activity requires interaction with PCNA, a DNA polymerase d cofactor, and chromatin. Importantly, p21 is an inhibitor of PCNA activity. As PCNA and DNA polymerase d are known to be required for parvoviral rolling hairpin replication, we hypothesized that MVM must target p21 for depletion to allow for PCNA activity, which is required for viral replication. p21 mutants that were unable to interact with either CRL4Cdt2 or PCNA were stable during MVM infection, suggesting that p21 needed to interact with both the E3 ubiquitin ligase and PCNA for degradation during MVM infection. We next constructed p21 mutants that were resistant to ubiquitination, by mutating the seven lysine residues in p21 to arginine, and maintained or lost their ability to interact with PCNA. Importantly, a stable p21 mutant that interacted with PCNA resulted in the significant depletion of MVM replication whereas a stable p21 mutant that no longer interacted with PCNA had no effect on replication. Taken together, this data suggests that MVM co-opts a cellular E3 ubiquitin ligase to target the CDK inhibitor p21 for degradation, which is required to allow the PCNA activity that MVM needs for efficient replication. To induce a pre-mitotic G2/M cell cycle block, MVM depletes the key mitotic cyclin, cyclin B1, which is preceded by the loss of its encoding RNA. We next sought to determine how MVM programs the depletion of cyclin B1 RNA. Initial studies indicated a loss of cyclin B1 RNA between 18 and 24 hours post-infection in a NS2-independent manner. This loss of RNA and protein was seen in both human and murine cells lines, suggesting that programming the depletion of cyclin B1 is a critical hallmark of MVM infection. Interestingly, the viral mechanism which targets cyclin B1 could overcome the effects of an exogenous DNA damaging agent known to induce cyclin B1 levels. The stability of cyclin B1 RNA during MVM infection is comparable to doxorubicin-treated cells, while the production of nascent cyclin B1 RNA is substantially depleted. Importantly, the chromatin landscape of the cyclin B1 promoter during MVM infection was consistent with an open conformation, yet significantly lower levels of RNA polymerase II (RNA pol II) were found to occupy the cyclin B1 gene. The NF-Y transcription factor and B-myb, a component of MuvB-B-Myb (MMB) complex, were both found to bind the cyclin B1 promoter during infection. However, the key G2/M transcription factor, FoxM1, was found to occupy the cyclin B1 promoter at significantly lower levels in MVM infected cells compared to mock- or doxorubicin-treated cells. FoxM1, which requires hyperphosphorylation to activate its transcriptional activity, was found to have lower levels of phosphorylation during MVM infection, compared to mock- or doxorubicin-treated cells. Reconstitution of FoxM1 to the cyclin B1 promoter, via catalytically inactive Cas9 fused to the FoxM1 transactivation domain, upregulated cyclin B1 RNA and protein during MVM infection. Taken together, these results suggest that MVM prevents the activation and binding of the critical transcription factor FoxM1 to the cyclin B1 promoter, thereby reducing RNA pol II transcriptional activity and the production of nascent cyclin B1 RNA and its encoded protein, ultimately facilitating establishment of a pre-mitotic G2/M block. An important RNA-binding protein, HuR, known to regulate two cyclins important for MVM infection, cyclin A and cyclin B1, was also investigated. We observed the predominantly nuclear HuR was heavily relocalized to the cytoplasm during MVM infection. Ectopic expression of NS1, but not NS2, resulted in the cytoplasmic relocalization of HuR in a Crm1-independent manner. Importantly, siRNA knockdown of HuR during MVM infection resulted in a further reduction of cyclin B1 protein, but not cyclin B1 RNA. This data suggested that HuR may promote the translation of specific RNAs during MVM infection. HuR was also shown to bind MVM RNA. While more work needs to be undertaken to fully understand the ramifications of this interaction, HuR is likely to be an important regulator of MVM RNA stability or translation. Taken together, these observations suggest that MVM expertly modulates the DDR and other components of the cellular machinery to ensure an environment conducive to facilitation of viral replication. To establish this environment, MVM targets several core cellular mechanisms including proteasome-mediated protein degradation, RNA transcription, RNA translation and subcellular localization of cellular proteins.