ICP27-dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0

Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD 50 ) for neurovirulence in mice following intracerebral inoculation about 10 3 -fold lower than the LD 50 of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo. Herpes simplex virus 1 (HSV-1) virions, like those of other herpesviruses, consist of three morphologically distinct structures: the nucleocapsid, containing the linear doublestranded DNA viral genome, which encodes at least 84 viral proteins, in an icosahedral capsid; the tegument, a proteinaceous layer surrounding the nucleocapsid; and the envelope, a host cellderived lipid bilayer with viral glycoproteins enclosing the nucleocapsid and tegument (55). The tegument consists of a large number of proteins, including at least 26 viral proteins (41). Tegument proteins are released into the cytoplasm upon the fusion of the virion envelope with the host cell membrane and therefore may function to establish an environment for effective initiation of very early infection. However, the roles of only a few tegument proteins, including the immediate-early gene transactivator VP16, encoded by the UL48 gene, and the virion host shutoff protein VHS, encoded by the UL41 gene (55), in very early infection have been established. Large amounts of newly synthesized tegument proteins have also been shown to modulate the host cellular environment and to regulate various viral replication processes later in infection (28).VP22, encoded by the UL49 gene, is one of the major HSV-1 tegument proteins, and its amino acid sequence is conserved in the subfamily Alphaherpesvirinae. It appears that the functional requirements of VP22 homologues in the viral life cycle differ among alphaherpesviruses. For example, the VP22 homologues of Marek's disease virus serotype 1 and varicella-zoster virus are considered to...

Section: Characterization Of a Ul49-null Mutant Virusmentioning

confidence: 99%

Herpes Simplex Virus 1 VP22 Regulates Translocation of Multiple Viral and Cellular Proteins and Promotes Neurovirulence

Tanaka

Kato

Satoh

et al. 2012

“…The inhibitory function was mapped to the carboxyl-terminal half of ICP27 (57,58). In more recent studies, Lengyel et al noted that leptomycin B causes massive export of ICP4 and ICP0 from the nucleus to the cytoplasm (24). They also reported that resistance to leptomycin B maps to residues 21 to 63 in the amino-terminal domain of ICP27 (24).…”

Section: Vol 82 2008mentioning

confidence: 99%

“…In more recent studies, Lengyel et al noted that leptomycin B causes massive export of ICP4 and ICP0 from the nucleus to the cytoplasm (24). They also reported that resistance to leptomycin B maps to residues 21 to 63 in the amino-terminal domain of ICP27 (24). Interestingly, the drug has no such effect in cells infected with leptomycin B-resistant mutants.…”

Section: Vol 82 2008mentioning

confidence: 99%

Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the U_L41 Gene

Kalamvoki

Roizman

2008

In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the U L 41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. In an earlier study concerning the redistribution of the IP3 receptor I in infected cells, we noted that the infected cell protein no. 4 (ICP4) is translocated into the cytoplasm in cells infected with mutant viruses lacking glycoprotein E or U L 41, the gene encoding the virion host shutoff protein (vhs) (18). This was an unexpected finding. ICP4 is one of several major regulatory proteins encoded by herpes simplex virus 1 (HSV-1). The protein acts both as a repressor and as a transactivator. Studies of temperature-sensitive mutants have shown that ICP4 is required throughout the replicative cycle of the virus (5, 6). Unlike ICP0, which late in infection with wild-type virus is translocated to the cytoplasm, ICP4 is thought to be a nuclear protein and in fact resides exclusively in the nucleus in wild-type virus-infected cells. This report centers on the role of gE, gI, and U L 41 proteins in the localization of ICP4 and ICP0 proteins. Relevant to this report are the following.(i) In wild-type virus-infected cells, there is not significant colocalization of ICP0 and ICP4. ICP0 initially colocalizes with the ND10 structure (12, 30, 31), fills the nucleus, and eventually, between 9 and 12 h after infection, is present in a diffuse form entirely in the cytoplasm (16,19,20,28). Export of ICP0 to the cytoplasm is blocked by exposure of cells 2 h after infection to proteasomal inhibitor MG132 (13,28). A striking feature of the translocation process is that exposure of infected cells to MG132 after ICP0 has been exported to the cytoplasm results in the relocation of ICP0 to the nucleus (28).ICP0 is also exported to the cytoplasm in cells infected with mutants lacking ICP4 (⌬ICP4) (16,28). Unlike the situation with wild-type virus-infected cells, ICP0 is translocated at earlier times after infection and forms small dense structures dispersed throughout the cytoplasm or arranged around the nucleus. In the presence of MG132, added late after infection, ICP0 is retained in the cytoplasm in similar small dense structures that also contain proteasomal components (28). Relevant to this report is the observation that a D199A substitution in ICP0 prevents the translocation of ICP0 to the cytoplasm (52,53). This observation suggests that the translocation of ICP0 is an active process involving an interactive partner.Last, ICP0 and ICP4 have been reported to colocalize in the cytoplasm in ce...

“…ICP27 is one of the best-characterized mRNA export factors that have been shown to favor viral RNA export and to retain cellular transcripts in the nucleus. ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich nuclear export signal (16,27) via a CRM1-dependent pathway (17). Interestingly, ICP27 and UL84 share many of the same functional characteristics associated with regulatory proteins.…”

Section: Discussionmentioning

confidence: 99%

Nucleocytoplasmic Shuttling of Human Cytomegalovirus UL84 Is Essential for Virus Growth

Gao

Kagele

Smallenberg

et al. 2010

Human cytomegalovirus (HCMV) UL84 is a multifunctional protein that is the proposed initiator for lytic viral DNA synthesis. Recently it was shown that UL84 displays nucleocytoplasmic shuttling. The role of shuttling in lytic DNA replication and virus growth is unknown. We now show that expression of the nonshuttling UL84 mutant failed to complement oriLyt-dependent DNA replication in the transient assay under conditions where core replication and ancillary proteins were expressed under the control of their native promoters. However, constitutive expression of the core replication proteins, along with the nonshuttling UL84 mutant, resulted in efficient oriLyt amplification, suggesting that shuttling may contribute to the activity of one of the auxiliary replication proteins. A recombinant HCMV bacterial artificial chromosome plasmid (BACmid) expressing the nonshuttling UL84 mutant (NS84 BAC) was defective for production of infectious virus. Quantitative PCR showed that NS84 BAC had decreased accumulation of viral DNA in both cellular and supernatant samples. Analysis of the accumulation of select viral mRNAs showed no difference in total cellular mRNA accumulation for IE2, IRS1, TRS1, UL102, UL105, and UL75 in cells transfected with the NS84 BAC. However, examination of cytoplasmic RNA and subcellular localization of IRS1 revealed a decrease in IRS1 mRNA accumulation and displaced protein localization, strongly suggesting that UL84 facilitated the localization of IRS1 mRNA to the cytoplasm. RNA pulldown assays showed that UL84 interacted with IRS1 mRNA. These results indicate that nucleocytoplasmic shuttling is essential for virus growth and strongly suggest that UL84 is responsible for localization of at least one virus-encoded transcript, IRS1 mRNA.Human cytomegalovirus (HCMV) is widely distributed in the human population, with a majority of adults eventually becoming infected. The HCMV genome is a 229-kb linear double-stranded DNA (dsDNA) molecule that encodes approximately 200 genes (5, 34). During productive infection, HCMV gene expression occurs in three main phases: immediate-early, early, and late (11). Lytic viral DNA synthesis requires both the cis-acting origin of lytic DNA replication (oriLyt) (1, 2, 12, 21) and a set of trans-acting viral proteins (23,24).UL84 is essential for virus replication and growth (31, 35) and is one of the noncore proteins required for origin-dependent DNA replication in a transient replication assay (23, 28). UL84 interacts with oriLyt within the region that contains RNA/DNA hybrid and stem-loop structures and CCAAT/ enhancer binding protein ␣ (C/EBP␣) binding motifs (7,14). The mechanism for initiation of HCMV DNA replication is proposed to be via a UL84 direct interaction with specific structures within oriLyt, and it is suggested that it could recruit other viral and/or cellular factors (4,14). UL84 can interact directly with the HCMV regulatory protein IE2, which is the major transcription-activating protein of HCMV (29). UL84 also interacts and colocalizes with UL44, ...