Nuclear and Mitochondrial Localization Signals Overlap within Bovine Herpesvirus 1 Tegument Protein VP22

Abstract

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.VP22 is a major component of the herpesvirus tegument, a viral compartment located between the capsid and envelope. As is true for many viral tegument proteins, the function of VP22 in viral replication and infection is yet to be elucidated. Bovine herpesvirus 1 (BHV-1) 1 VP22 is dispensable for viral growth in cell culture, but deletion of VP22 from the virus genome delays virus growth and greatly reduces virus yield (1). A herpes simplex virus-1 mutant expressing a truncated form of VP22 has been characterized (2). Interestingly a deletion mutant of VP22 is avirulent in infected cattle, suggesting that VP22 is an important virulence factor and may play a modulatory role in BHV-1 in vivo infection (3).Although VP22 has no "classical" nuclear localization signal (NLS), it accumulates in the nucleus of infected cells by an unknown mechanism. In the nucleus, VP22 associates with a variety of viral and cellular factors. For example, VP22 binds VP16, an important viral transactivator of early gene expression, and is considered to regulate the function of VP16 (4). VP22 also interacts with template-activating factor I, a chromatin-remodeling protein. Template-activating factor I promotes an ordered transfer of histones to naked DNA, and binding of VP22 to template-activating factor I may regulate the function of template-activating factor I and interfere with nucleosomal deposition of the virus genome in early infection (5). We previously reported that BHV VP22 also binds nucleosomes, interfering with acetylation of histone H4 (6), suggesting a possible regulatory role of VP22 in virus infection. Interestingly expression of VP22 in transfected cells up-regulates the expression ratio of Bax/Bcl-2 inducing apoptosis (7). In addition, herpes simplex virus VP22 can bind and traffic mRNA t...

Cell Cycle Modulation by Marek’s Disease Virus: The Tegument Protein VP22 Triggers S-Phase Arrest and DNA Damage in Proliferating Cells

“…However, among the characteristics of VP22, we can speculate that its capacity to interact with chromatin and histones might participate to those processes. Interactions of VP22 protein with nucleosomes were previously demonstrated for the BoHV-1-encoded VP22, which physically interacts with nucleosome-associated histones and thereby causes an impaired acetylation of histone H4 [21], [56]. In addition, for MDV-VP22, the regions allowing interaction with heterochromatin were previously defined [17].…”

“…However, the precise role of VP22 in MDV replication and MD pathogenesis remains unclear. Notably, the functional significance of the VP22 nuclear distribution is still unknown, even if previous reports on VP22 encoded by alphaherpesviruses evoke a possible regulatory function of VP22 within nuclei [17], [20], [21], [22].…”

Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1

“…That an NLS sequence is able to mediate nuclear retention has been demonstrated for other proteins. For instance, the NLS of bovine herpesvirus 1 tegument protein VP22 can bind to histones to promote its nuclear accumulation [43]. The NLSs of many transcription factors are located within the DNA-binding domain that is considered to be a potential nuclear retention signal [21].…”

Metabolic Maturation of Auditory Neurones in the Superior Olivary Complex

“…The specificity of all primary antibodies used has been previously published and the relevant publications are indicated for the respective antibodies. The primary antibodies used were: chicken anti-Map2 (1∶1000, Neuromics, CH22103 [45], [63]), mouse anti-ATPase (1∶1000, DSHB, a5 [64], [65]), rabbit anti-synapsin (1∶100, SySy, 106 002 [66]), rabbit anti-GLUT3 (1∶100, abcam, ab41525 [62], [67]) and mouse anti-mitochondria (1∶500, abcam, ab3298 [68]). The anti-mitochondria antibody was raised against a non-glycosylated protein component of the mitochondrial membrane obtained from a partially purified mitochondrial preparation.…”