scholarly journals Human Cytomegalovirus Essential Tegument Protein pp150 is Amenable to Targeting by Sequence Specific Peptides and ELP-conjugates

2021 ◽  
Author(s):  
Dipanwita Mitra ◽  
Mohammad H Hasan ◽  
Gene L. Bidwell, III ◽  
Ritesh Tandon
Keyword(s):  
Human Cytomegalovirus ◽  
3D Modeling ◽  
Capsid Proteins ◽  
Tegument Protein ◽  
Virus Growth ◽  
Nuclear Egress ◽  
Binding Interface ◽  
Carrier Molecule ◽  
Inhibitory Potential

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of final steps in virion maturation. Earlier studies indicated that three pp150nt (N terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B) and extend towards small capsid proteins atop nearby major capsid proteins forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. To enhance the in-vivo inhibitory potential and bioavailability of pep-CR2, we conjugated it with a carrier molecule (elastin like polypeptide (ELP)). The ELP-pep-CR2 conjugate was expressed in E.coli and purified. Upon treatment with ELP-pep-CR2, HCMV showed significant titer reductions with no significant impact on cell viability. These results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor and can be developed into an effective antiviral.

scholarly journals Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2277
Author(s):  
Dipanwita Mitra ◽  
Mohammad H. Hasan ◽  
John T. Bates ◽  
Gene L. Bidwell ◽  
Ritesh Tandon
Keyword(s):  
Cytomegalovirus Infection ◽  
Capsid Proteins ◽  
Tegument Protein ◽  
Animal Testing ◽  
Mouse Cytomegalovirus ◽  
Mcmv Infection ◽  
Virus Growth ◽  
Nuclear Egress ◽  
Binding Interface ◽  
Specific Peptide

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150–capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

scholarly journals Daxx-Mediated Accumulation of Human Cytomegalovirus Tegument Protein pp71 at ND10 Facilitates Initiation of Viral Infection at These Nuclear Domains

2002 ◽  
Vol 76 (15) ◽  
pp. 7705-7712 ◽  
Author(s):  
Alexander M. Ishov ◽  
Olga V. Vladimirova ◽  
Gerd G. Maul
Keyword(s):  
Human Cytomegalovirus ◽  
Coiled Coil ◽  
Immediate Early ◽  
Productive Infection ◽  
Tegument Protein ◽  
Knockout Mutant ◽  
Nuclear Site ◽  
Immediate Early Proteins ◽  
Nuclear Domains

ABSTRACT Human cytomegalovirus (HCMV) starts immediate-early transcription at nuclear domains 10 (ND10), forming a highly dynamic immediate transcript environment at this nuclear site. The reason for this spatial correlation remains enigmatic, and the mechanism for induction of transcription at ND10 is unknown. We investigated whether tegument-based transactivators are involved in the specific intranuclear location of HCMV. Here, we demonstrate that the HCMV transactivator tegument protein pp71 accumulates at ND10 before the production of immediate-early proteins. Intracellular trafficking of pp71 is facilitated through binding to a coiled-coil region of Daxx. The C-terminal domain of Daxx then interacts with SUMO-modified PML, resulting in the deposition of pp71 at ND10. In Daxx-deficient cells, pp71 does not accumulate at ND10, proving in vivo the necessity of Daxx for pp71 deposition. Also, HCMV forms immediate transcript environments at sites other than ND10 in Daxx-deficient cells, and so does the HCMV pp71 knockout mutant UL82−/− in normal cells. This result strongly suggests that pp71 and Daxx are essential for HCMV transcription at ND10. Lack of Daxx had the effect of reducing the infection rate. We conclude that the tegument transactivator pp71 facilitates viral genome deposition and transcription at ND10, possibly priming HCMV for more efficient productive infection.

scholarly journals Resistance of Human Cytomegalovirus to the Benzimidazole l-Ribonucleoside Maribavir Maps to UL27

2003 ◽  
Vol 77 (21) ◽  
pp. 11499-11506 ◽  
Author(s):  
Gloria Komazin ◽  
Roger G. Ptak ◽  
Brian T. Emmer ◽  
Leroy B. Townsend ◽  
John C. Drach
Keyword(s):  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Resistance Mutation ◽  
Open Reading Frames ◽  
Cosmid Library ◽  
Wild Type Virus ◽  
Wild Type ◽  
Nuclear Egress ◽  
The One

ABSTRACT 1-(β-d-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.

scholarly journals Atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp150

Science ◽  
2017 ◽  
Vol 356 (6345) ◽  
pp. eaam6892 ◽  
Author(s):  
Xuekui Yu ◽  
Jonathan Jih ◽  
Jiansen Jiang ◽  
Z. Hong Zhou
Keyword(s):  
Human Cytomegalovirus ◽  
Atomic Structure ◽  
Rational Design ◽  
Capsid Proteins ◽  
Tegument Protein ◽  
A Genome ◽  
Hiv Aids

Herpesviruses possess a genome-pressurized capsid. The 235-kilobase genome of human cytomegalovirus (HCMV) is by far the largest of any herpesvirus, yet it has been unclear how its capsid, which is similar in size to those of other herpesviruses, is stabilized. Here we report a HCMV atomic structure consisting of the herpesvirus-conserved capsid proteins MCP, Tri1, Tri2, and SCP and the HCMV-specific tegument protein pp150—totaling ~4000 molecules and 62 different conformers. MCPs manifest as a complex of insertions around a bacteriophage HK97 gp5–like domain, which gives rise to three classes of capsid floor–defining interactions; triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. HCMV-specific strategies include using hexon channels to accommodate the genome and pp150 helix bundles to secure the capsid via cysteine tetrad–to-SCP interactions. Our structure should inform rational design of countermeasures against HCMV, other herpesviruses, and even HIV/AIDS.

scholarly journals Gammaherpesvirus Tegument Protein ORF33 Is Associated with Intranuclear Capsids at an Early Stage of the Tegumentation Process

2015 ◽  
Vol 89 (10) ◽  
pp. 5288-5297 ◽  
Author(s):  
Sheng Shen ◽  
Xing Jia ◽  
Haitao Guo ◽  
Hongyu Deng
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Early Stage ◽  
Lytic Replication ◽  
Capsid Proteins ◽  
Tegument Protein ◽  
Virus Propagation ◽  
Murine Gammaherpesvirus ◽  
Tegument Proteins ◽  
Nuclear Egress

ABSTRACTHerpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study thede novolytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.IMPORTANCEMorphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.

scholarly journals Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress

2016 ◽  
Vol 90 (16) ◽  
pp. 7109-7117 ◽  
Author(s):  
Bernadette M. DeRussy ◽  
Molly T. Boland ◽  
Ritesh Tandon
Keyword(s):  
Human Cytomegalovirus ◽  
Pseudorabies Virus ◽  
Capsid Assembly ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Virus Growth ◽  
Virus Maturation ◽  
Nuclear Egress ◽  
Multiple Regions

ABSTRACTHuman cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth, but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids and interacts with the components of the nuclear egress complex (NEC), namely, pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 serves as an important link between nucleocapsid maturation and nuclear egress.IMPORTANCEHCMV causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely, pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point toward an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

scholarly journals Comparison between Human Cytomegalovirus pUL97 and Murine Cytomegalovirus (MCMV) pM97 Expressed by MCMV and Vaccinia Virus: pM97 Does Not Confer Ganciclovir Sensitivity

2000 ◽  
Vol 74 (22) ◽  
pp. 10729-10736 ◽  
Author(s):  
Markus Wagner ◽  
Detlef Michel ◽  
Peter Schaarschmidt ◽  
Bianca Vaida ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Human Cytomegalovirus ◽  
Recombinant Vaccinia Virus ◽  
Murine Cytomegalovirus ◽  
Virus Growth ◽  
Recombinant Vaccinia ◽  
Vaccinia Viruses ◽  
In Vivo Testing ◽  
Bacterial Plasmid

ABSTRACT The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056–7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

An Attempt at Biological Control of Blossom Blight of Rose Caused by Botrytis cinerea Using some Local Trichoderma spp. Strains

2020 ◽  
Vol 55 (1) ◽  
pp. 27-34
Author(s):  
G. Zadehdabagh ◽  
K. Karimi ◽  
M. Rezabaigi ◽  
F. Ajamgard
Keyword(s):  
Botrytis Cinerea ◽  
Mycelial Growth ◽  
Limiting Factor ◽  
Dual Culture ◽  
Trichoderma Spp ◽  
Khuzestan Province ◽  
Inhibitory Potential ◽  
Cut Rose

The northern of Khuzestan province in Iran is mainly considered as one of the major areas of miniature rose production. Blossom blight caused by Botrytis cinerea has recently become a serious limiting factor in rose production in pre and post-harvest. In current study, an attempt was made to evaluate the inhibitory potential of some local Trichoderma spp. strains against B. cinerea under in vitro and in vivo conditions. The in vitro results showed that all Trichoderma spp. strains were significantly able to reduce the mycelial growth of the pathogen in dual culture, volatile and non-volatile compounds tests compared with control, with superiority of T. atroviride Tsafi than others. Under in vivo condition, the selected strain of T. atroviride Tsafi had much better performance than T. harzianum IRAN 523C in reduction of disease severity compared with the untreated control. Overall, the findings of this study showed that the application of Trichoderma-based biocontrol agents such as T. atroviride Tsafi can be effective to protect cut rose flowers against blossom blight.

scholarly journals Spanning the gap: unraveling RSC dynamics in vivo

2021 ◽  
Author(s):  
Heinz Neumann ◽  
Bryan J. Wilkins
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Binding Mode ◽  
Binding Modes ◽  
Binding Domains ◽  
Binding Interface ◽  
The Many ◽  
And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

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