A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Strang, Blair L.; Bender, Brian J.; Sharma, Madhu; Pesola, Jean M.; Sanders, Rebecca; Spector, Deborah H.; Coen, Donald M.

doi:10.1128/jvi.01379-12

Cited by 20 publications

(21 citation statements)

References 56 publications

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“…To examine the importance of individual residues in the groove and cavity during HCMV replication, four substitutions in UL50, which have varying importance for heterodimerization in vitro, were introduced into a GFPencoding BAC derived from HCMV strain AD169, pBADGFP (35), or modifications of this BAC, in which sequences encoding an FLAG tag have been fused to UL53 coding sequences (8). Electroporation of BACs containing WT UL50 and mutants V52A and S125A that had 5-to 10-fold reductions in UL53 binding in vitro (Table 1) into human foreskin fibroblasts (HFFs) resulted in a spreading infection, with increasing numbers of GFP-expressing cells showing cytopathic effect, and production of infectious virus.…”

Section: Significancementioning

confidence: 99%

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Leigh

Sharma

Mansueto

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.H erpesviruses encompass a large family of infectious agents, including important veterinary and human pathogens (1). Among the latter is human cytomegalovirus (HCMV), which can cause serious disease, particularly in immunocompromised individuals and newborns (2). Despite the importance of HCMV in these medically vulnerable populations, currently available treatment options suffer from issues with toxicities, drug resistance, and/or pharmacokinetics (2, 3), motivating the identification of new drug targets.All herpesviruses of mammals, birds, and reptiles undergo a remarkable process known as nuclear egress as part of the viral lifecycle. It is generally accepted that, after assembly in the nucleus, the viral nucleocapsid undergoes envelopment to cross the inner nuclear membrane (INM) followed by deenvelopment to cross the outer nuclear membrane, resulting in release into the cytoplasm for continuation of the virion maturation process (4). Nuclear egress is orchestrated by a highly conserved, heterodimeric nuclear egress complex (NEC), which recruits one or more protein kinases to disrupt the nuclear lamina, permitting access of nucleocapsids to the INM, where the NEC induces budding of the nucleocapsid into the perinuclear space (5-13). In HCMV, the NEC is comprised of UL50, which is an INM protein, and UL53, which is a nucleoplasmic protein that is brought to the INM by its interaction with UL50. These two proteins and their murine CMV (MCMV) homologues, M50 and M53, are essential for replication and nuclear egress (8, 14-17) of ...

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Section: Significancementioning

confidence: 99%

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Leigh

Sharma

Mansueto

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…DNA was isolated from infected cells using a NucleoSpin tissue kit (Macherey-Nagel) according to the manufacturer's instructions. Viral genomes were quantified with a primer pair (pp549s and pp812as) specific for UL83 (21), and the number of viral genomes was normalized to the number of cellular copies of adipsin (22). Values for unknown samples were determined on the basis of standard curves of known copy numbers of UL83 (pcDNAUL83; a kind gift from Jeremy Kamil, Louisiana State University) and adipsin (from uninfected cell DNA).…”

Section: Cells and Virusesmentioning

confidence: 99%

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

Polachek

Moshrif

Franti

et al. 2016

J Virol

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High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCEThere is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell. I dentification of factors encoded by the cell that are required for virus replication can illuminate important features of virus-host interactions and identify novel drug targets for therapeutic intervention. Stages of productive human cytomegalovirus (HCMV) replication take place in both the nucleus and the cytoplasm (1). After replication of the viral DNA genome in the nucleus, newly synthesized viral genomic DNA is packaged into nascent capsids in the nucleus. These capsids then bud through the nuclear membrane and, after accessing a viral assembly compartment in the cytoplasm (2), undergo a process of secondary envelopment in the cytoplasm in which capsids gain a lipid bilayer before exiting the cell (1). Many of these processes require the function of cellular factors, a number of which are unknown.High-throughput small interfering RNA (siRNA) screening has been a successful strategy to identify cellular factor...

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“…A nucleolus has a tripartite structure in which the fibrillar centre is surrounded by a dense fibrillar component (containing the majority of nucleolin found in nucleoli) that is surrounded by the granular component. In HCMVinfected cells, nucleoli remain intact (Strang et al, 2012c), and visualization of nucleolin in HCMV-infected cells suggests changes in the size of nucleoli upon infection (Strang et al, 2012a). Also, striking changes to nucleolar composition and architecture occur, as, following infection, nucleolin levels in nucleoli increase and nucleolin accumulates at the periphery of nucleoli (Strang et al, 2012a) (Fig.…”

Section: Structures Associated With Hcmv Replication: Nucleoli Cajalmentioning

confidence: 99%

“…However, it is unclear if the reported nucleolar caps are actually associated with nucleoli. Furthermore, analysis of nucleoli in HCMV-infected cells by electron microscopy does not suggest the presence of nucleolar caps (Strang et al, 2012c). It is unknown whether HCMV infection stimulates p53-mediated nucleolar stress responses, which include specific changes to the protein translational profile of the cell (Boulon et al, 2010).…”

Section: Structures Associated With Hcmv Replication: Nucleoli Cajalmentioning

confidence: 99%

“…It is possible that HCMV also utilizes these cellular cargo transporters to facilitate capsid movement. Recently, it has been reported that mutation of HCMV UL84 leads to a defect in virus replication associated with a defect in nuclear egress of capsids and the clustering of capsids around nucleoli (Strang et al, 2012c). This would suggest that UL84 is, directly or indirectly, involved in the efficient movement of HCMV capsids within the nucleus.…”

Section: Exit Of Capsids From the Nucleus: The Nuclear Laminamentioning

confidence: 99%

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Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

Strang

2015

Journal of General Virology

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In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virus-host interactions in the nucleus. IntroductionThe betaherpesvirus human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that severely affects immunocompromised and immunodeficient populations (Mocarski et al., 2007). Like all herpesviruses, HCMV undergoes either productive or latent infection. During productive infection, infectious virus is produced, while in latent infection the virus becomes largely transcriptionally quiescent (Mocarski et al., 2007). Like other herpesviruses, many events that are critical for productive HCMV replication take place within the nucleus. These include essential steps in viral replication, such as: the transcriptional cascade of immediate-early (IE), early and late viral RNA transcripts, synthesis of viral DNA and the production of DNA-containing capsids (Mocarski et al., 2007). Compared with the prototype human herpesvirus, the alphaherpesvirus herpes simplex virus (HSV), certain features of productive HCMV infection are not well characterized. However, it is clear that several subnuclear structures are involved in HCMV genome replication and virus-host interactions. Uncovering the roles of these structures has led to significant advances in our understanding of HCMV biology. This review summarizes the involvement of several viral and cellular subnuclear structures in productive HCMV infection. The participation of each structure in HCMV replication and virus-host interactions is described from entry of the viral genome into the nucleus to the egress of viral capsids through the nuclear membrane. The data discussed highlight recent advances in our understanding of subnuclear structure function, introduce viral and cellular factors associated with subnuclear structures and indicate what questions have yet to be answered.Entry of the HCMV genome into the nucleus: the nuclear pore complex, nuclear speckles, promyelocytic leukaemia protein nuclear bodies and pp150 bodies HCMV genome entry into the nucleus is poorly defined but may be similar to movement of the HSV DNA genome through the nuclear pore complex (NPC) (Kobiler et al., 2012) (Fig. 1a). HCMV capsid ...

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A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Cited by 20 publications

References 56 publications

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

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