Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient spread by the extracellular route but not for direct cell-to-cell spread

Human cytomegalovirus (HCMV) encodes multiple G protein-coupled receptor (GPCR) homologues, including pUS27, pUS28, pUL33, and pUL78. To explore the function of pUS27, we constructed pUS27-deficient derivates of two clinical isolates of HCMV. BFX-GFPstopUS27 is a FIX variant with a single base pair change in the US27 open reading frame, generating a stop codon that ablates accumulation of the GPCR homologue, and TB40/E-mCherrydlUS27 lacks the entire US27 coding region. BFX-GFPstopUS27 generated 10-fold less extracellular progeny in fibroblasts, and TB40/E-mCherrydlUS27 exhibited a similar defect in endothelial cells. The pUS27-deficient FIX derivative produced normal quantities of viral DNA and viral proteins tested, and a late virion protein was appropriately localized to the cytoplasmic assembly zone. After infection at a low multiplicity with wild-type FIX virus, neutralizing antibody reduced the accumulation of intracellular viral DNA and intracellular virions, as would be expected if the virus is limited to direct cell-to-cell spread by neutralization of extracellular virus. In contrast, the antibody had little effect on the spread of the BFX-GFPstopUS27 virus. Further, after infection at a low multiplicity, the pUS27-deficient TB40/E virus exhibited a growth defect in endothelial cells, where the clinical isolate normally generates extracellular virus, but the TB40/E derivative exhibited little defect in epithelial cells, where the wild-type virus does not produce extracellular virus. Thus, mutants lacking pUS27 rely primarily on direct cell-to-cell spread, and we conclude that the viral GCPR homologue acts at a late stage of the HCMV replication cycle to support spread of virus by the extracellular route.     

Efficient incorporation of tegument proteins pUL46, pUL49, and pUS3 into pseudorabies virus particles depends on the presence of pUL21

The mature virion of the alphaherpesvirus pseudorabies virus (PrV) contains a minimum of 31 structural proteins which are recruited into the virus particle by a network of protein-protein interactions which is only incompletely understood. We show here that deletion of the tegument protein pUL21 resulted in a drastic decrease in the incorporation of the pUL46, pUL49, and pUS3 tegument components into mature virions. Moreover, the attenuated PrV strain Bartha (PrV-Ba), which, among other defects, carries mutations in pUL21, also fails to package pUL46, pUL49, and pUS3 efficiently. By the reconstitution of wild-type pUL21 expression to PrV-Ba and the transfer of mutated PrV-Ba pUL21 into wild-type PrV, we demonstrate that this phenotype is due to the mutated pUL21.     

Analysis of viral and cellular factors influencing herpesvirus-induced nuclear envelope breakdown

Herpesvirus nucleocapsids are translocated from their assembly site in the nucleus to the cytosol by acquisition of a primary envelope at the inner nuclear membrane which subsequently fuses with the outer nuclear membrane. This transport through the nuclear envelope requires homologs of the conserved herpesviral pUL31 and pUL34 proteins which form the nuclear egress complex (NEC). In its absence, 1,000-fold less virus progeny is produced. We isolated a UL34-negative mutant of the alphaherpesvirus pseudorabies virus (PrV), PrV-ΔUL34Pass, which regained replication competence after serial passages in cell culture by inducing nuclear envelope breakdown (NEBD) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 85:8285-8292, 2011). To test whether this phenotype is unique, passaging experiments were repeated with a UL31 deletion mutant. After 60 passages, the resulting PrV-ΔUL31Pass replicated similarly to wild-type PrV. Ultrastructural analyses confirmed escape from the nucleus via NEBD, indicating an inherent genetic disposition in herpesviruses. To identify the mutated viral genes responsible for this phenotype, the genome of PrV-ΔUL34Pass was sequenced and compared to the genomes of parental PrV-Ka and PrV-ΔUL34. Targeted sequencing of PrV-ΔUL31Pass disclosed congruent mutations comprising genes encoding tegument proteins (pUL49, pUL46, pUL21, pUS2), envelope proteins (gI, pUS9), and protease pUL26. To investigate involvement of cellular pathways, different inhibitors of cellular kinases were tested. While induction of apoptosis or inhibition of caspases had no specific effect on the passaged mutants, roscovitine, a cyclin-dependent kinase inhibitor, and U0126, an inhibitor of MEK1/2, specifically impaired replication of the passaged mutants, indicating involvement of mitosis-related processes in herpesvirus-induced NEBD.     

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.     

Emerin is hyperphosphorylated and redistributed in herpes simplex virus type 1-infected cells in a manner dependent on both UL34 and US3

Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKCdelta that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.     

Identification of functional domains within the essential large tegument protein pUL36 of pseudorabies virus

Proteins of the capsid proximal tegument are involved in the transport of incoming capsids to the nucleus and secondary envelopment after nuclear egress. Homologs of the essential large capsid proximal tegument protein pUL36 are conserved within the Herpesviridae. They interact with another tegument component, pUL37, and contain a deubiquitinating activity in their N termini which, however, is not essential for virus replication. Whereas an internal deletion of 709 amino acids (aa) within the C-terminal half of the alphaherpesvirus pseudorabies virus (PrV) pUL36 does not impair its function (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006), deletion of the very C terminus does (J. Lee, G. Luxton, and G. A. Smith, J. Virol. 80:12086-12094, 2006). For further characterization we deleted several predicted functional and structural motifs within PrV pUL36 and analyzed the resulting phenotypes in cell culture and a mouse infection model. Extension of the internal deletion to encompass aa 2087 to 2981 exerted only minor effects on virus replication but resulted in prolonged mean survival times of infected mice. Any additional extension did not yield viable virus. Deletion of an N-terminal region containing the deubiquitinating activity (aa 22 to 248) only slightly impaired viral replication in cell culture but slowed neuroinvasion in our mouse model, whereas a strong impairment of viral replication was observed after simultaneous removal of both nonessential domains. Absence of a region containing two predicted leucine zipper motifs (aa 748 to 991) also strongly impaired virus replication and spread. Thus, we identify several domains within the PrV UL36 protein, which, though not essential, are nevertheless important for virus replication.     

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17⁺ did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.     

Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein.     

Cyclin A Degradation by Primate Cytomegalovirus Protein pUL21a Counters Its Innate Restriction of Virus Replication

Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus'' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.     

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.     

Nuclear envelope breakdown can substitute for primary envelopment-mediated nuclear egress of herpesviruses

Herpesvirus nucleocapsids assemble in the nucleus but mature to infectious virions in the cytoplasm. To gain access to this cellular compartment, nucleocapsids are translocated to the cytoplasm by primary envelopment at the inner nuclear membrane and subsequent fusion of the primary envelope with the outer nuclear membrane. The conserved viral pUL34 and pUL31 proteins play a crucial role in this process. In their absence, viral replication is strongly impaired but not totally abolished. We used the residual infectivity of a pUL34-deleted mutant of the alphaherpesvirus pseudorabies virus (PrV) for reversion analysis. To this end, PrV-ΔUL34 was serially passaged in rabbit kidney cells until final titers of the mutant virus PrV-ΔUL34Pass were comparable to those of wild-type PrV. PrV-ΔUL34Pass produced infectious progeny independently of the pUL34/pUL31 nuclear egress complex and the pUS3 protein kinase. Ultrastructural analyses demonstrated that this effect was due to virus-induced disintegration of the nuclear envelope, thereby releasing immature and mature capsids into the cytosol for secondary envelopment. Our data indicate that nuclear egress primarily serves to transfer capsids through the intact nuclear envelope. Immature and mature intranuclear capsids are competent for further virion maturation once they reach the cytoplasm. However, nuclear egress exhibits a strong bias for nucleocapsids, thereby also functioning as a quality control checkpoint which is abolished by herpesvirus-induced nuclear envelope breakdown.     

Meiotic nuclear divisions in budding yeast require PP2A(Cdc55)-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.     

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (Mastl^NULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in Mastl^NULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, Mastl^NULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of Mastl^NULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.     

Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production: inactivating NLS1, NLS2, or both affects replication to strikingly different extents

Scaffolding proteins of spherical prokaryotic and eukaryotic viruses have critical roles in capsid assembly. The primary scaffolding components of cytomegalovirus, called the assembly protein precursor (pAP, pUL80.5) and the maturational protease precursor (pPR, pUL80a), contain two nuclear localization sequences (NLS1 and NLS2), at least one of which is required in coexpression experiments to translocate the major capsid protein (MCP, pUL85) into the nucleus. In the work reported here, we have mutated NLS1 and NLS2, individually or together, in human cytomegalovirus (HCMV, strain AD169) bacmid-derived viruses to test their effects on virus replication. Consistent with results from earlier transfection/coexpression experiments, both single-mutant bacmids gave rise to infectious virus but the double mutant did not. In comparisons with the wild-type virus, both mutants showed slower cell-to-cell spread; decreased yields of infectious virus (3-fold lower for NLS1(-) and 140-fold lower for NLS2(-)); reduced efficiency of pAP, pPR, and MCP nuclear translocation (sixfold lower for NLS1(-) and eightfold lower for NLS2(-)); increased amounts of a 120-kDa MCP fragment; and reduced numbers of intranuclear capsids. All effects were more severe for the NLS2(-) mutant than the NLS1(-) mutant, and a distinguishing feature of cells infected with the NLS2(-) mutant was the accumulation of large, UL80 protein-containing structures within the nucleus. We conclude that these NLS assist in the nuclear translocation of MCP during HCMV replication and that NLS2, which is unique to the betaherpesvirus UL80 homologs, may have additional involvements during replication.     

Pseudorabies Virus Fast Axonal Transport Occurs by a pUS9-Independent Mechanism

Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein pUS9 promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.     

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.