Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus

We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.     

Targeting of the gene product encoded by ORF UL56 of human cytomegalovirus into viral replication centers

The highly conserved DNA-binding protein pUL56 of human cytomegalovirus (HCMV) was found to be predominantly localized throughout the nucleus as well as in viral replication centers of infected cells. The latter localization was abolished by phosphono acetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL56 co-localized in replication centers alongside pUL112-113 and pUL44 at late times of infection. By co-immunoprecipitations, a direct interaction with pUL44, a protein of the replication fork, was detected. These results showed for the first time that HCMV pUL56 is localized in viral replication centers, implicating that DNA replication is coupled with packaging.     

Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 microM. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.     

Interactions among the bHLH domains of the proteins encoded by theEnhancer of split andachaete-scute gene complexes ofDrosophila

TheEnhancer of split andachaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis inDrosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.     

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.     

Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid

Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.     

Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile(135) of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.     

Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts

Background

The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice.

Results

The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains.

Conclusion

The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.     

Relationship between autophosphorylation and phosphorylation of exogenous substrates by the human cytomegalovirus UL97 protein kinase

Human cytomegalovirus encodes an unusual protein kinase, UL97, which is a member of the HvU(L) family of protein kinases encoded by diverse herpesviruses. UL97 is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. It has previously been concluded that phosphorylation of UL97 is essential for its phosphorylation of ganciclovir. We examined the relationship between autophosphorylation of UL97 and its activity on exogenous substrates. Glutathione S-transferase-UL97 fusion protein purified from insect cells was found to be already partially phosphorylated, but neither extensive autophosphorylation nor phosphatase treatment meaningfully altered the time course of its phosphorylation of the exogenous substrate, histone H2B. Sequencing and mass spectrometric analyses of (32)P-labeled tryptic peptides of the UL97 fusion protein identified nine sites of autophosphorylation, all within the first 200 residues of the protein, outside of conserved protein kinase subdomains. A peptide corresponding to the N-terminal UL97 segment that was most extensively autophosphorylated was readily phosphorylated by UL97, confirming that fusion protein sequences are not required for phosphorylation at this site. Deletion mutants lacking at least the first 239 residues exhibited drastically reduced autophosphorylation (<5%) but retained near-wild-type H2B phosphorylation activity. Baculoviruses expressing these mutants efficiently directed the phosphorylation of ganciclovir in insect cells. Taken together, these results identify the autophosphorylation sites of a herpesvirus protein kinase and show that autophosphorylation of UL97 is not required for phosphorylation of exogenous substrates.     

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR

Phosphorylation of G-protein-coupled receptors (GPCRs) by GRKs and subsequent recruitment of beta-arrestins to agonist-occupied receptors serves to terminate or attenuate signaling by blocking G-proteins from further interaction with the receptors. Human cytomegalovirus encodes a GPCR termed US28 that is homologous to the human chemokine family of GPCRs but differs from the cellular receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is constitutively active, mechanisms that regulate this activity are unknown. We provide evidence that US28 is constitutively phosphorylated by GRKs in cells and that in consequence, beta-arrestin 2 is localized to the plasma membrane. Deletion of the carboxyl terminal 40 amino acids in US28 generates a receptor that is severely impaired in its ability to become phosphorylated and recruit beta-arrestin and accordingly demonstrates increased inositol phosphate signaling. This result indicates that the carboxyl terminus of US28 contains an important signaling regulatory region and mutational analysis deleting carboxyl terminal serines identified serine 323 as a critical residue within this region. In addition, overexpression of wild type GRK5 leads to hyperphosphorylation of US28 that results in a decrease of inositol phosphate accumulation. These results are consistent with the hypothesis that GRK phosphorylation and recruitment of beta-arrestin to the US28 viral GPCR attenuates signaling to the traditional Galphaq-stimulated inositol phosphate pathway. Finally, in contrast to the results with inositol phosphate signaling, we provide evidence that the US28 carboxyl-terminal phosphorylation sites and beta-arrestin-interacting domain are required for maximal activation of the p38 mitogen-activated protein kinase. Taken together, these results indicate that US28 interacts with these important regulatory proteins to control multiple aspects of signal transmission. Understanding the regulation of viral GPCRs by GRKs and beta-arrestins will provide important new insights into not only aspects of viral pathogenesis but also basic mechanisms of receptor signaling.     

Interactions among the bHLH domains of the proteins encoded by theEnhancer of split andachaete-scute gene complexes ofDrosophila

TheEnhancer of split andachaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis inDrosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.     

The application of high-performance liquid chromatography for the resolution of proteins encoded by the human adenovirus type 2 cell transformation region

The human adenovirus 2 (Ad2) transformation genes are located in early region E1a (map position (mp) 1.3–4.5) and E1b (mp 4.6–11.2) on the linear duplex Ad2 DNA genome of M_r 23 × 10⁶ (viral DNA is divided into 100 map units). E1b codes for three major proteins of apparent molecular weights 53,000 (53K), 19K, and 20K; smaller quantities of 21K, 22K, and 23K proteins that are related to 53K are also synthesized in Ad2-infected cells. Because the resolution and purification of these Ad2 candidate transformation proteins proved very difficult by conventional protein purification methods, the applicability of high-performance liquid chromatography (HPLC) methodology was examined. Starting with a crude cytoplasmic S100 fraction of Ad2-infected human cells, the resolution of the Ad2 E1b-coded 19K, 20K, 21K, 22K, and 23K proteins by reverse-phase HPLC using a C₈ column and a linear 0–60% 1-propanol gradient in 0.5 m pyridine formate was achieved, E1b proteins purified under these conditions retained their immunological reactivity. By anion-exchange HPLC using a linear 10 mm to 1 m NaCl gradient in 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.6, the same five Ad2 E1b-coded 19K–23K proteins were separated, with improved resolution of the 19K protein. Based on these findings, protocols for the extensive purification of the E1b-19K and E1b-20K proteins have been developed. These results illustrate the potential of HPLC methodology for the rapid purification of biologically interesting proteins from complex cellular mixtures of proteins.     

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12bp, with apparent dissociation constants in the nanomolar range for DNAs >18bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4±1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity.     

Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery

The retinoblastoma (RB) family consists of three genes, RB1, RBL1, and RBL2, that code for the pRb, p107, and pRb2/p130 proteins, respectively. All these factors have pivotal roles in controlling fundamental cellular mechanisms such as cell cycle, differentiation and apoptosis. The founder and the most investigated RB family protein is pRb, which is considered to be the paradigm of tumor suppressors. However, p107 and pRb2/p130 clearly display a high degree of structural and functional homology with pRb. Interestingly, these factors were first identified as physical targets of the Adenovirus E1A oncoprotein. Indeed, RB family proteins are the most important and widely investigated targets of small DNA virus oncoproteins, such as Adenovirus E1A, human papillomavirus E7 and Simian virus 40 large T antigen. By interacting with pRb and with other RB family members, these oncoproteins neutralize their growth suppressive properties, thus stimulating proliferation of the infected cells, de‐differentiation, and resistance to apoptosis. All these acquired features strongly favor the rise and selection of immortalized and mutation‐prone cells, leading to a higher propensity in undergoing transformation. Our present work aims to illustrate and delve into these protein–protein interactions. Considering that these viral oncoproteins are dispensable for normal cellular functions, they can create “oncogene addiction” in the infected/transformed cells. This makes the possibility to dismantle these interactions extremely attractive, thus promoting the development of highly specific smart molecules capable of targeting only the infected/transformed cells that express these viral factors. J. Cell. Physiol. 228: 285–291, 2013. © 2012 Wiley Periodicals, Inc.