The flexible loop of the human cytomegalovirus DNA polymerase processivity factor ppUL44 is required for efficient DNA binding and replication in cells

Dipartimento di Ematologia e Scienze Oncologiche L.A. Seragnoli, Università Degli Studi di Bologna, Italy.
Journal of Virology (Impact Factor: 4.65). 08/2009; 83(18):9567-76. DOI: 10.1128/JVI.00669-09
Source: PubMed

ABSTRACT Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP(174)]), which has been proposed to be directly involved in ppUL44's interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Deltaloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Deltaloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Deltaloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Deltaloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.

Download full-text


Available from: Gregory S Pari, Aug 24, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.
    Journal of Virology 10/2010; 84(24):12589-98. DOI:10.1128/JVI.01394-10 · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxy-terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.
    Journal of Virology 11/2010; 84(21):11563-8. DOI:10.1128/JVI.01033-10 · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.
    Journal of Biological Chemistry 02/2011; 286(16):14335-51. DOI:10.1074/jbc.M110.210302 · 4.57 Impact Factor
Show more