As one of the most ubiquitous pathogens that has a worldwide distribution, herpes simplex virus type 1 (HSV-1) infects the respiratory or oropharyngeal mucosa and causes a variety of diseases ranging from mild illness to severe life-threatening infections such as herpes keratoconjunctivitis and herpes encephalitis. The primary infection in the mucosal epithelial cells is lytic while the virus gains access to the termini of sensory neurons and establishes life-long latent infections, which can transmit to the peripheral sites after reactivation and lead to recurrent lesions in the vicinity of the primary infected area. During productive infection, the linear, double stranded HSV-1 genome of approximately 152 kb encodes at least 84 proteins which are separated into three subsets known as immediate-early (IE), early (E), and late (L) proteins according to the sequential expression of the corresponding genes. More than half of these genes are poorly understood functionally [12, 13, 24] and identified as dispensable proteins for viral replication at least in some cell lines [1, 2]. These dispensable gene products are, however, demonstrated to be required for virus growth and pathogenesis in experimental animal systems [18, 20, 25, 31].
One such nonessential protein, UL4, has been identified and classified as a true late (γ2) gene product , but no functions have yet been assigned. Homologues of the UL4 gene are present in other members of the Alphaherpesviruses, such as the HSV-2 gene UL4 , the varicella-zoster virus gene 56 , the equine herpesvirus-1 gene 58 , the pseudorabies virus (PrV) gene UL4  and the bovine herpesvirus-1 gene (BHV-1) UL4 . Conservation of the gene suggests that it may play a certain role in the viral life cycle. Recent investigation has revealed that the PrV UL4 protein is a non-structural protein which is important for virus yields, release of mature virions, and enhancement of virion formation but it is not essential for PrV replication in vitro or in vivo .
ICP22 is an IE protein that enhances the expression of a subset of γ2 proteins. The UL4 protein has been identified as a putative nuclear protein for exhibiting co-localization with ICP22 in small, dense nuclear bodies in infection , suggesting the intriguing possibility of partial function overlap of ICP22 and the UL4 protein, and the UL4 protein may play certain roles related with ICP22 in the context of the small, dense nuclear compartment associated with late gene transcription. Furthermore, the late protein UL3 and UL20.5 are subsequently identified as the components of the discrete, dense nuclear spots [21, 36]. Since deletion of ICP22 resulted in a decreased accumulation of UL3 and, to a lesser extent, of UL4, and as neither UL3 nor UL4 localizes to the dense nuclear structures, with additional evidence showing that ICP22 by itself is sufficient to form small, dense nuclear bodies in transfection assay without other viral proteins, it has been suggested that co-localization of UL3 and UL4 is directed by ICP22 rather than by the interaction of the UL3 and UL4 proteins . However, our recent systematic study showed determinately that both UL3 and UL20.5 are targeted to small, dense nuclear bodies by direct interaction with ICP22, whereas UL4 co-localizes with ICP22 through its direct interaction with UL3 but neither UL20.5 nor ICP22, and there is no interaction between UL3 and UL20.5 . We also observed that UL4 is not a genuine nuclear protein as it localizes to sub-cytoplasmic structures when expressed alone. In addition, UL4 re-localized UL3 to the cytoplasm structure when co-expressed, while the subcellular distribution of UL4 was not altered when co-expressed with ICP22, but UL3 was redistributed in the nuclear foci identical to the subcellular localization pattern of ICP22, indicating that UL3 is the intermediate to bring UL4 to ICP22 and is required for the localization of UL4 in the nuclear compartment. Interestingly, the interaction between UL4 and UL3 took place in the cytoplasm where UL4 was located when expressed alone, prompting further investigation of the subcellular localization of UL4, which would be helpful to dig deeper not only into the interaction between UL3 and UL4, but also the role and mechanism of that interaction complex during the proliferation of HSV-1.
Active export of a protein from the nucleus depends on the presence of a specific nuclear export signal (NES) [10, 16]. To date three types of NES have been identified, for instance the ~10 amino acid leucine-rich NES initially identified in protein kinase inhibitor (PKI) and HIV-1 Rev, the M9 sequence of hnRNP A1, and a 24 amino acid stretch found in hnRNPK. In addition, an increasing number of viral proteins, including HIV-1 Rev [16, 26, 27], HSV-1 UL47 , the RSV matrix protein  and BHV-1 protein BICP27  have been found to contain a leucine-rich NES sequence, enabling the protein to be exported out of the nucleus. Chromosomal region maintenance 1 (CRM1; exportin 1), identified as an export receptor that recognizes NES directly, is responsible for the export of NES-containing proteins through the nuclear pore complex (NPC) using RanGTP [34, 37]. The pharmacological compound leptomycin B (LMB) directly interacts with CRM1 and blocks NES-mediated protein export.
In this study, we used living cell fluorescence microscopy, which has been extensively applied  and developed in our laboratory [11, 39, 41] to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HSV UL4 protein combined with enhanced yellow fluorescent proteins (EYFP) to identify the molecular determinants for subcellular localization of UL4; the molecular mechanisms underlying the nuclear export of UL4 was also elucidated.
Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein
- Received Date: 04 August 2011
- Accepted Date: 05 September 2011
Abstract: The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), the nuclear export signals (NES) of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 (CRM1)-dependent manner involving RanGTP hydrolysis.