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Title: Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus}
Article:
Abstract:
Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus( HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.
Introduction:
Control of the cell division cycle by cyclins, cyclin-dependent kinases( CDKs) and CDK inhibitors( CKIs) is fundamental for proliferation, development and homeostasis of multicellular organisms[1, 2]. To reprogram the cell cycle for their own benefit, viral pathogens have evolved, or acquired from their host, genes and sequence motifs facilitating direct interaction with the cyclin-CDK protein network[3]. Herpesviruses are particularly well suited for multifaceted interactions with the cell cycle machinery, owing to the large coding capacity of their genomes. The repertory of herpesviral cell cycle regulators comprises on the one hand factors leading to constitutive activation of the cell cycle. This is exemplified by the β and γ-herpesviral orthologs of cyclins[4] and CDKs[5], which release CDK substrate phosphorylation from the control of cellular cyclins and CKIs[6, 7]. On the other hand, herpesviruses target cellular cyclin-CDK activity to arrest the cell cycle at stages conducive to virus replication[8]. A recent example is the UL21a gene product( pUL21a) of human cytomegalovirus( HCMV), which is required to block DNA synthesis and mitotic entry of infected cells[9, 10]. Like CKIs of the Cip/Kip family( p21, p27, p57), pUL21a contains a high affinity RXL-type cyclin binding motif but is only a poor CDK substrate[10]. In contrast to CKIs, however, pUL21a does not act as a stoichiometric inhibitor of cyclin-CDK complexes but specifically recruits cyclin A( also referred to as cyclin A2) for proteasomal degradation[9, 10]. Viral interactions with the cell cycle are not necessarily unidirectional. HCMV encodes a second RXL-type cyclin A-binding protein, pp150( also referred to as pUL32), that is neither an activator nor an inhibitor of the cell cycle but is itself subject of cyclin A-CDK-dependent regulation[11]. PP150 enters the host cell as part of the HCMV virion and blocks de novo viral gene expression in a cyclin A and CDK-dependent manner[12, 13]. In fibroblasts and other permissive cell types, this mechanism restricts the onset of viral replication to the G0/G1 phase of the cell cycle where cyclin A expression is low or absent. S/G2 cells, though, do not abrogate but only delay infection as pp150-mediated repression is relieved once cells loose cyclin A protein after cell division[14, 15]. Animal CMVs, including chimpanzee CMV, the closest relative of HCMV, lack RXL sequence motifs in their pp150 homologues and accordingly initiate viral gene expression independent of the cell cycle position at the time of infection[11, 16]. Thus, it is yet unclear what function, if any, the pp150-dependent restriction serves in the context of productive HCMV infection. Here, we show that the pp150-RXL motif, alone, is dispensable for efficient viral growth. However, genetic disruption of both pp150 and pUL21a-RXL motifs dramatically enhances the mitotic phenotype and growth defect of a pUL21a-RXL single mutant virus. Thus, the cyclin A antagonist pUL21a and the cyclin A sensor pp150 are part of a virus-host interface, that functions as a fail-safe system securing undisturbed HCMV replication under non-mitotic conditions.
Discussion:
HCMV encodes two cyclin A interacting proteins, the cyclin A-CDK substrate pp150 and the cyclin A destabilizing module pUL21a. Here we show that both proteins act synergistically, together constituting a control circuit required for the synchronization of viral replication with the cell division cycle. The tegument protein pp150 senses the cellular cyclin A status at the beginning of infection and restricts the onset of IE gene expression to the G0/G1 phase, where cyclin A2-CDK is inactive. The early gene product pUL21a maintains the status of low cyclin A2-CDK activity by targeting cyclin A for proteasomal degradation. Thus, pp150-mediated restriction of viral gene expression in S/G2 phase has a genuine function in the productive replication cycle of HCMV instead of being merely an inevitable by-product of a silencing mechanism that contributes to establishing quiescent infection in undifferentiated cells[11]. The pp150-dependent restriction to cyclin A-negative cells appears important enough to justify a significant delay of virus gene expression and replication after infection of proliferating cells( Figs 2E and 3C). This is particularly remarkable in view of the high overall growth rates of a cyclin A sensor-less pp150-RXL mutant virus in both G1 and G2 cell cycle compartments( Figs 1C and 3C). In that respect, pp150 mutant HCMV behaves like animal CMVs, which lack a pp150-cyclin A interface and therefore can efficiently replicate in S/G2 cells[11, 16]. Taken together, this may point to a model where cyclin A sensing by pp150 alone is not absolutely required for productive HCMV infection but has been developed to strengthen and support cell cycle synchronization by pUL21a. Because pUL21a is expressed with early kinetics and therefore not available during the first hours of infection[31], a pre-synchronization step by a protein like pp150, delivered by the incoming virion, makes sense and keeps lytic gene expression in safe distance from G2/M transition, giving pUL21a time to install a stable cell cycle arrest in interphase. If this presynchronization is missing the pUL21a function is not sufficient, or is not present early enough, to tightly inhibit mitotic entry in every infected cell( Figs 2F, 2G and 3B). The described synchronization strategy of HCMV is in striking analogy to how another human pathogen, human papilloma virus( HPV) coordinates its replication with the host cell cycle. Just as HCMV, many HPV strains encode two RXL motif containing cyclin A interactors, E1 and E1˄E4[32, 33]. The early protein E1 is a DNA helicase that, due to its cyclin A-CDK-dependent nuclear localization, is only able to initiate viral DNA synthesis in its CDK-phosphorylated form[33, 34]. This makes sense from the viewpoint of HPV given that this small DNA virus in contrast to HCMV heavily depends on the cellular DNA replication machinery, which is only available and active at times of high cyclin A-CDK activity. The late protein E1˄E4 re-localizes cyclin A-CDK to the cytoplasm, thereby preventing the onset of mitosis[35, 36]. Thus, both HCMV and HPV have evolved two layers of cyclin A interaction. The first layer consists of cyclin A-sensitive CDK substrates that confine the start of viral replication to the most suitable cell cycle phase—G1 in case of HCMV, S phase in case of HPV. The second layer consists of potent, negative regulators of cyclin A-CDK that provide stable conditions for virus growth by arresting the cell cycle in interphase( HCMV: G1 arrest, HPV: G2 arrest). It is fascinating, that two only distantly related viruses like HCMV and HPV have developed such similar strategies to best synchronize their life cycle with that of virally favorable phases of the cell cycle for reaching highest efficacy and efficiency of viral replication. Although HCMV, HPV and many other viruses have evolved sophisticated mechanisms to prevent mitotic entry in productively infected cells[37], it is important to note that in other contexts, such as virus entry or persistence, viruses use mitosis to their own advantage. Herpesviruses as well as papillomaviruses encode mitotic chromosome tethering factors to maintain latent viral genomes episomally in dividing cells[38–41]. Papillomaviruses and select retroviruses require the nuclear envelope breakdown in mitosis for nuclear import of viral genomes[42]. Why then mitosis presents a problem when it occurs in the middle of the productive replication cycle of HCMV? Certainly, the global shutdown of gene transcription[43] and mRNA translation[44] in mitosis would seem to be counterproductive for a virus that hijacks the host cell machinery to achieve maximum replication. Also, structural changes like the nuclear envelope breakdown are potentially harmful for the functional integrity of viral replication and assembly compartments[10]. However, if mitosis would represent only a short and transient interruption in the replication cycle of HCMV, the virus could possibly cope with such perturbations. But, HCMV, as many viruses, encode potent inhibitors of the anaphase promoting complex( APC/C) to stabilize substrates of this E3 ubiquitin ligase that play a role in viral replication[45–48]. Due to the essential function of APC/C in mitosis, productively infected cells cannot simply traverse through this cell cycle phase—they become arrested at the metaphase-anaphase transition[10]. Another aspect is that HCMV expresses DNA damaging enzymes[49, 50] and subverts cellular DNA repair[51, 52]. It is unclear, as yet, to what extent virus-induced DNA breaks contribute to the chromosomal fragmentation visible in mitotic cells( S7 Fig). However, unrepaired DNA damage and prolonged metaphase arrest are known to cause cell death by mitotic catastrophe[53, 54] and, though HCMV is a master in preventing premature cell death during interphase[55], it is evidently not equally prepared to protect productively infected cells in mitosis.
Efficient virus replication depends on continuous , uninterrupted supply with metabolites and replication factors from the host cell . This is difficult to achieve in actively dividing cells , especially for a slowly replicating virus like HCMV , a widespread pathogen of major medical importance in immunocompromised patients . To ensure that viral replication is not disturbed by cell division , HCMV has developed a twofold strategy of cyclin A targeting and avoidance . First , HCMV employs the viral cyclin A substrate pp150 to synchronize the onset of replication with G1 , a cell cycle phase of low cyclin A expression . Then , HCMV expresses the cyclin A destabilizing factor pUL21a to maintain the G1 cell cycle state until the successful release of virus progeny . While this strategy is based on two viral proteins , a cyclin A sensor and effector , it relies on one and the same type of cyclin A interaction motif , making HCMV vulnerable to binding site disruption .