Identification of Nuclear and Nucleolar Localization Signals of Pseudorabies Virus (PRV) Early Protein UL54 Reveals that Its Nuclear Targeting Is Required for Efficient Production of PRV

Abstract

The pseudorabies virus (PRV) early protein UL54 is a homologue of herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein that is essential for HSV-1 infection. In this study, the subcellular localization and nuclear import signals of PRV UL54 were characterized. UL54 was shown to predominantly localize to the nucleolus in transfected cells. By constructing a series of mutants, a functional nuclear localization signal (NLS) and a genuine nucleolar localization signal (NoLS) of UL54 were for the first time identified and mapped to amino acids 61 RQRRR 65 and 45 RRRRGGRGGRAAR 57 , respectively. Additionally, three recombinant viruses with mutations of the NLS and/or the NoLS in UL54 were constructed based on PRV bacterial artificial chromosome (BAC) pBecker2 to test the effect of UL54 nuclear targeting on viral replication. In comparison with the wild-type virus, a recombinant virus harboring an NLS or NoLS mutation of UL54 reduced viral production to different extents. However, mutations of both the NLS and NoLS targeted UL54 to the cytoplasm in recombinant virus-infected cells and significantly impaired viral replication, comparable to the UL54-null virus. In addition, a virus lacking the NLS or the NoLS displayed modest defects in viral gene expression and DNA synthesis. However, deletion of both the NLS and the NoLS resulted in severe defects in viral gene expression and DNA synthesis, as well as production of infectious progeny. Thus, we have identified a classical NLS and a genuine NoLS in UL54 and demonstrate that the nuclear targeting of UL54 is required for efficient production of PRV.

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

“…This demonstrates that structural dynamics can alter the signals presented by the protein to the nuclear transport machinery. Numerous viruses use classic NLSs for nuclear targeting of viral proteins (28)(29)(30).…”

Preparation and characterization of an antiserum against truncated UL54 protein of pseudorabies virus

“…However, UL54 only located to the nucleus when PK-15 cells were infected with PRV at high MOI (MOI = 1) at all time points examined, implying that the subcellular localization of UL54 also correlates with the virus titer . Indeed, the correlation between viral gene expression and virus MOI has been described before and multiplicity-dependent growth phenotype has also been observed for several viruses, including PRV, HSV-1, human cytomegalovirus, African swine fever virus and adenovirus (Nevins, 1981;Cai and Schaffer, 1992;Chen and Silverstein, 1992;Moore et al, 1998;Bresnahan and Shenk, 2000;Everett et al, 2004;Fang et al, 2004;Li et al, 2011b). Therefore, the different subcellular localization of UL54 suggests that UL54 perhaps plays an important role in the gene expression, efficient PRV production or some other processes in the course of viral replication cycle.…”

“…Moreover, the truncated UL54 contains both of the nuclear localization signal (NLS) and nucleolar localization signal (NoLS), which have been described before, thus makes the prepared antiserum a good tool for further studying the functions of NLS and NoLS of UL54 or/and full-length UL54 (Li et al, 2011b).…”

“…Different intracellular localizations may reflect different functions of viral proteins, and the intracellular localizations of viral proteins may also vary at different times after the infection (Feng, 2002;Cai et al, 2009a). Early studies demonstrated that UL54 is a nuclear protein, which can localize exclusively to the nucleolus in the absence of other viral proteins, whereas the native UL54 in PRV-infected cells is delocalized from the nucleolus to the nucleus in a structure that might resemble the viral replication compartment during infection (Monier et al, 2000;Calle et al, 2008;Li et al, 2011b). In line with previous studies (Li et al, 2011a,b), our results here showed that the localization of UL54 was changed from nucleolus to the entire nucleus as the virus replicated in PK-15 cells at low MOI, suggesting that the subcellular localization pattern of UL54 is associated with viral replication cycle at low MOI (MOI = 0.1) (Li et al, 2011a,b).…”

Identification of Synonymous Codon Usage Bias in the Pseudorabies Virus UL31 Gene

“…Regarding the role of UL31 gene product played in the herpesvirus life cycle, herpes simplex virus 1 (HSV-1) UL31 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36) and Epstein-Barr virus (EBV) BFLF2 (37)(38)(39), the homologue of PRV UL31, have been extensively studied; however, the precise function propers of PRV UL31 gene, as well as its codon usage bias is weakly understood.…”