CMV Pp28

What is CMV pp28 and where is it located in the virion structure?

CMV pp28 is a 190-amino-acid myristylated phosphoprotein that constitutes one of the most abundant components of the viral tegument. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. It is encoded by UL99, the last open reading frame positioned within a family of 3′-coterminal transcripts that share the same polyadenylation site. In the tegument, pp28 forms a critical structural component that contributes to the integrity of the mature virion .

What is the expression pattern of pp28 during HCMV infection?

Pp28 is expressed as a true late protein during HCMV infection, meaning it is synthesized only after the onset of viral DNA replication. This timing places pp28 expression in the final stages of the viral replication cycle, coinciding with virion assembly processes. The expression pattern directly correlates with its function in the late stages of virion morphogenesis, particularly during the envelopment process .

What post-translational modifications occur on pp28?

The pp28 protein undergoes two significant post-translational modifications:

• Myristoylation: Addition of a myristoyl group to the glycine residue following the translation initiation methionine. This modification is essential for membrane association.

• Phosphorylation: Multiple sites on pp28 are phosphorylated, though the exact phosphorylation pattern and its functional significance require further characterization.

These modifications, particularly myristoylation, are critical for the proper localization and function of pp28 in infected cells. When myristoylation is prevented through mutation of the glycine residue, the intracellular distribution and detergent solubility properties of pp28 are significantly altered .

What methods are effective for studying pp28 multimerization?

Researchers studying pp28 multimerization commonly employ the following methodology:

• Metabolic labeling with [35S]Met-Cys for 10 minutes

• Chase with medium containing unlabeled Met/Cys and cycloheximide

• Cell solubilization in TBS containing 0.1% NP-40

• Preclearing of lysates with normal goat serum and staphylococcal Cowan I bacteria

• Separation on 5-40% linear sucrose gradients (34,000 rpm for 20h in Beckman SW41 rotor)

• Gradient fractionation and immunoprecipitation

• Analysis by SDS-PAGE

This approach allows researchers to track newly synthesized pp28 and monitor its assembly into multimeric complexes over time, providing insights into the dynamics of tegument protein assembly .

How can researchers effectively determine pp28 membrane association?

Membrane association of pp28 can be evaluated using Triton X-114 (TX114) partitioning assays:

• Harvest infected cells 6 days post-infection

• Solubilize in TX114 at 4°C

• Clarify the detergent phase (containing membranes)

• Warm solution to 30°C and partition into detergent and aqueous phases via low-speed centrifugation

• Analyze fractions by immunoblotting

Using this method, researchers have demonstrated that the majority of pp28 partitions into the detergent phase, confirming its membrane association. Similar treatment of gradient-purified extracellular virions indicates that virion pp28 is also predominantly associated with the viral envelope .

What expression systems are recommended for studying pp28 in isolation?

To study pp28 function independent of other viral proteins, researchers have successfully used:

• Recombinant retrovirus expression systems:

  • Construction of retroviruses (e.g., Retropp28WT and RetroHA-pp28)

  • Infection of human fibroblasts in presence of Polybrene (15 μg/ml)

  • Sequential infections for achieving higher expression levels

• Bacterial expression systems:

  • E. coli-derived recombinant protein containing immunodominant regions

  • GS-4B Sepharose-affinity purification

  • Formulation in 50mM Tris-HCl pH 7.2, 1mM EDTA and 50% glycerol

These systems allow investigation of pp28 properties in the absence of other viral components, helping researchers understand its intrinsic characteristics versus those dependent on viral context .

What happens to HCMV replication when pp28 is absent?

Studies using pp28-null mutant viruses have revealed that pp28 is absolutely essential for HCMV replication. Two constructed mutants—BADsubUL99 (substitution mutant) and BADpmUL99 (point mutant)—were profoundly defective for growth in normal fibroblasts, with no detectable infectious virus production after infection.

When pp28 is absent:

• Viral DNA synthesis proceeds normally

• Late viral proteins are expressed at normal levels

• Large numbers of tegument-associated capsids accumulate in the cytoplasm

• These capsids fail to acquire an envelope

These findings demonstrate that pp28 plays a critical role in the final envelopment of the HCMV virion in the cytoplasm, a step essential for the production of infectious virus particles .

Which cellular compartment does pp28 localize to during infection, and why is this significant?

In infected cells, pp28 localizes to a cytoplasmic compartment derived from the Golgi apparatus, specifically where the virus buds into vesicles to acquire its final membrane. When expressed in the absence of other viral proteins, pp28 localizes to the ER-Golgi-intermediate compartment (ERGIC), which interfaces with both the ER and Golgi apparatus.

This localization is significant because:

• It confirms that HCMV tegument assembly includes a cytoplasmic phase

• It suggests that viral tegument protein interactions within the secretory pathway are critical for virion assembly

• It indicates that additional viral functions are required for the relocalization of pp28 to the cytoplasmic assembly compartment observed in HCMV-infected cells

The restriction of pp28 to the ERGIC in the absence of other viral proteins provides evidence that the assembly program of HCMV has a complex cytoplasmic phase involving interactions between tegument proteins and envelope proteins within the cellular secretory pathway .

How does myristoylation affect pp28 function in HCMV assembly?

Myristoylation plays a crucial role in pp28 function through several mechanisms:

• Membrane association: Myristoylation anchors pp28 to cellular membranes, as demonstrated by Triton X-114 partitioning assays

• Subcellular localization: The modification is required for proper targeting to the ERGIC and subsequently to the cytoplasmic assembly compartment

• Detergent solubility: Mutation of the myristoylation site alters the detergent solubility properties of pp28

• Assembly function: The membrane association facilitated by myristoylation is likely essential for pp28's role in facilitating the final envelopment of cytoplasmic capsids

Without myristoylation, pp28 cannot properly associate with membranes of the secretory pathway, preventing its participation in the envelopment process that occurs as capsids bud into vesicles to acquire their final envelope .

How does pp28 multimerization contribute to virion assembly?

Recent research suggests that pp28 forms multimeric complexes during HCMV assembly, which may serve as scaffolding structures that facilitate the organization of other tegument proteins. The multimerization process appears to be temporally regulated during infection, with early monomeric forms progressively assembling into larger complexes.

Experimental evidence for multimerization comes from sedimentation analysis in sucrose gradients, where pp28 can be detected in multiple fractions corresponding to different molecular weights. This suggests that pp28 exists in various oligomeric states within infected cells. The functional significance of these multimers likely relates to creating a protein network that bridges the capsid and envelope during the envelopment process .

What is the relationship between pp28 and other tegument proteins during assembly?

The assembly of the HCMV tegument involves a complex network of protein-protein interactions, with pp28 playing a central role. While the complete interaction network is still being elucidated, several key relationships have been identified:

• Pp28 likely interacts with other abundant tegument proteins such as pp65 and pp71

• These interactions may form a protein scaffold that facilitates the recruitment of additional tegument components

• The assembly process appears to be hierarchical, with certain interactions preceding others

Understanding these protein-protein interactions is critical for developing a comprehensive model of HCMV tegument assembly and potentially identifying new targets for antiviral intervention .

What are the comparative functions of pp28 homologs across different herpesviruses?

The UL99-encoded pp28 has homologs in other herpesviruses, most notably the UL11 protein of Herpes Simplex Virus (HSV). Comparative analysis reveals:

What challenges arise when creating pp28 mutant viruses, and how can they be overcome?

Creating pp28 mutant viruses presents several technical challenges:

• Essential nature of pp28: Since pp28 is essential for viral replication, null mutants cannot be propagated in standard cell cultures

  • Solution: Use complementing cell lines expressing pp28 (e.g., HFFpp28-3x, HFFpp28-8x, or HFFHApp28)

• Low efficiency of retroviral transduction:

  • Solution: Perform sequential infections (3-8 times) to achieve adequate pp28 expression levels

• Potential for recombination restoring wild-type sequence:

  • Solution: Careful screening of viral stocks by PCR and sequencing to confirm maintenance of mutations

• Distinguishing mutant phenotypes from effects on overlapping genes:

  • Solution: Create multiple types of mutations (substitution mutants versus point mutants) to ensure consistent phenotypes

What are the optimal conditions for detecting pp28 in immunoassays?

For reliable detection of pp28 in various immunoassay formats:

• Western blotting:

  • Use freshly prepared samples

  • Include appropriate reducing agents

  • Run controls with recombinant pp28 for size comparison

• ELISA:

  • The E. coli-derived recombinant protein containing CMV pp28 (UL99) immunodominant regions (amino acids 130-160) provides excellent specificity

  • 95% purity as determined by 10% PAGE is recommended for quantitative assays

  • Use sera from CMV-infected individuals as positive controls

• Sample storage:

  • Store pp28 protein below -18°C

  • Avoid freeze-thaw cycles to maintain antigenicity

  • For long-term storage, aliquot preparations in 50mM Tris-HCl pH 7.2, 1mM EDTA and 50% glycerol

How can researchers differentiate between the roles of pp28 myristoylation and phosphorylation in functional studies?

To dissect the distinct contributions of myristoylation versus phosphorylation to pp28 function:

• Site-directed mutagenesis approach:

  • Create G2A mutants to specifically block myristoylation

  • Generate serine/threonine to alanine mutants at known phosphorylation sites

  • Develop combination mutants affecting both modifications

• Biochemical inhibition:

  • Use myristoylation inhibitors (e.g., 2-hydroxymyristic acid) with appropriate controls

  • Apply phosphatase inhibitors or treatment with phosphatases in parallel experiments

• Analytical methods:

  • Employ TX114 partitioning to assess membrane association

  • Use phospho-specific antibodies to monitor phosphorylation status

  • Perform immunofluorescence microscopy to track localization changes

• Functional readouts:

  • Compare virus production using complementation assays

  • Examine ultrastructural details of virion assembly by electron microscopy

These approaches allow researchers to attribute specific functional aspects to each post-translational modification, providing a more nuanced understanding of how these modifications collaborate to support pp28's role in virion assembly .

What emerging technologies could advance our understanding of pp28 dynamics during infection?

Several cutting-edge approaches show promise for elucidating pp28 function:

• Live-cell imaging techniques:

  • Fluorescently tagged pp28 variants for real-time tracking

  • Super-resolution microscopy to visualize pp28 distribution at nanoscale resolution

  • FRAP (Fluorescence Recovery After Photobleaching) to study pp28 mobility

• Proximity labeling methods:

  • BioID or APEX2 fusions to identify proteins in close proximity to pp28

  • Temporal analysis of the pp28 interactome during infection progression

• Cryo-electron tomography:

  • Direct visualization of pp28 organization within the tegument layer

  • Structural analysis of assembly intermediates

These technologies would provide dynamic, high-resolution insights into pp28 behavior during the viral life cycle, moving beyond the static snapshots currently available .

How might understanding pp28 function contribute to novel antiviral strategies?

Given pp28's essential role in HCMV replication, it presents a promising target for antiviral development:

• Small molecule inhibitors:

  • Compounds targeting pp28 multimerization

  • Inhibitors of pp28-membrane association

  • Molecules disrupting pp28 interactions with other tegument proteins

• Peptide-based approaches:

  • Peptides mimicking critical pp28 interaction domains

  • Cell-penetrating peptides that compete for binding sites

• Host-directed therapeutics:

  • Compounds modulating the cellular compartments where pp28 functions

  • Inhibitors of enzymes responsible for pp28 post-translational modifications

Development of these strategies requires deeper structural and functional characterization of pp28, but could potentially yield antivirals with mechanisms distinct from current nucleoside analogs and polymerase inhibitors .