HVS (herpesvirus saimiri) is the prototype gamma-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using

Herpesvirus saimiri (HVS) ORF 57 is homologous to genes identified in all classes of herpesviruses.We have previously shown that ORF 57 encodes a multifunctional protein, responsible for both transactivation and repression of viral gene expression at a post-transcriptional level. This suggests that the ORF 57 protein shares some functional similarities with the herpes simplex virus IE63/ICP27 and Epstein-Barr virus Mta proteins. However, little is known about the functional domains responsible for the properties of ORF 57 due to the limited homology shared between these proteins. In this report, we have identified the functional domains responsible for transactivation and repression by the ORF 57 protein. We demonstrate that the carboxy terminus is required for ORF 57 transactivation, repression and an intense SC-35 nuclear spotting. This region contains two highly conserved motifs amongst its homologues, a zinc finger-like motif and a highly hydrophobic domain. We further show that the hydrophobic domain is required for transactivation and is also involved in nuclear localization of the ORF 57 protein, whereas the zinc finger-like domain is required for transactivation, repression and the intense SC-35 nuclear spotting.

Herpesvirus saimiri (HVS) is the prototype ␥-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human ␥-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin ␣ isoforms 1 and 5. In addition, the binding of ORF 57 to importin ␣ was mediated by the importin ␣ hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin ␣ into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.A number of viruses including herpes, adenovirus, influenza, and retroviruses replicate in the host cell nucleus. In order to complete their virus replication cycle, they have devised a number of mechanisms to transport viral nucleic acids into and out of the nucleus. In particular, a variety of viruses encode nucleocytoplasmic shuttle proteins, which specifically mediate the nuclear export of viral RNA. Such virally encoded proteins include human immunodeficiency virus type 1 (HIV-1) 1 Rev, herpes simplex virus type 1 (HSV-1) ICP27, influenza virus NEP, adenovirus E4orf6, and the herpesvirus saimiri (HVS) ORF 57 protein (1-6). Herpesvirus saimiri (HVS) is the prototype ␥-2 herpesvirus, or rhadinovirus (7), an increasingly important family of viruses due to the recent identification of the first human ␥-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus (8). Gene expression during HVS lytic replication is sequentially regulated and occurs in three main temporal phases: immediate-early, delayed-early, and late. The two major HVS transcriptional regulating proteins are encoded by the open reading frames (ORFs) 50 and 57 (9 -12).ORF 57 is a 52-kDa multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Transactivation of late viral genes by ORF 57 occurs independently of target gene promoter sequences and appears to be mediated at a post-transcriptional level (11). In addition to its transactivation properties, ORF 57 is responsible for repression of viral gene expression, which correlates with the presence of introns within the target gene (11-12). ORF 57 also redistributes both U2 and SC-35 splicing factors during an HVS infection into intense d...

The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 encoded protein of Kaposi's sarcomaassociated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localizes with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, the expression of HVS ORF73 in a stably transduced human carcinoma cell line, where the HVS genome persists as a non-integrated circular episome, has recently been shown. In this report, the characterization of the HVS ORF73 protein and the mapping of its functional domains are described. The results suggest that the HVS ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the amino terminus contains two functional nuclear localization signals, whereas the carboxy terminus is required for the distinctive speckled nuclear distribution pattern as observed with both the HVS and KSHV ORF73 proteins.

The herpesvirus saimiri (HVS) gene product encoded by ORF 57 shares limited C-terminal similarity with herpes simplex virus 1 ICP27, a protein that has been demonstrated to be involved in the inhibition of host-cell splicing and is responsible for the redistribution of components of the spliceosome. It has previously been shown that ORF 57 can either activate or repress viral gene expression by a post-transcriptional mechanism. Furthermore, repression of gene expression by ORF 57 is dependent on the presence of an intron within the target gene coding region. In this report, it is shown that HVS infection results in the redistribution of the SC-35 splicing factor in the infected cell nucleus. Furthermore, the redistributed SC-35 colocalized with the ORF 57 protein product and expression of the protein alone was sufficient to cause the redistribution of the spliceosome components. These results suggest that the mechanism by which ORF 57 downregulates expression of intron-containing genes involves the redistribution of the spliceosome complex.

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The herpesvirus saimiri open reading frame (ORF) 57 is homologous to genes identified in all classes of herpesviruses. It has previously been shown to regulate gene expression through a posttranscriptional mechanism. We demonstrate in this report that the expression of the ORF 57 protein leads to the cytoplasmic accumulation of glycoprotein B and capsid mRNAs. We also demonstrate that ORF 57 has the ability to specifically bind viral RNA transcripts. Utilizing an interspecies heterokaryon assay, we show that ORF 57 has the ability to shuttle between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology with nuclear export signals (NES) found in a number of proteins with the ability to shuttle between the nucleus and the cytoplasm. Moreover, we demonstrate that the ORF 57 NES enables the nuclear export of a heterologous protein and that mutation of the conserved leucine residues contained within the ORF 57 NES signal abrogates the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.