U
            <sub>S</sub>
            3 protein kinase of HSV-1 cycles between the cytoplasm and nucleus and interacts with programmed cell death protein 4 (PDCD4) to block apoptosis

Leptomycin B (LMB) is a highly specific inhibitor of CRM1, a cellular karyopherin-␤ that transports nuclear export signal-containing proteins from the nucleus to the cytoplasm. Previous work has shown that LMB blocks herpes simplex virus 1 (HSV-1) replication in Vero cells and that certain mutations in viral immediate early protein ICP27 can confer LMB resistance. However, little is known of the molecular mechanisms involved. Here we report that HSV-2, a close relative of HSV-1, is naturally resistant to LMB. To see whether the ICP27 gene determines this phenotypic difference, we generated an HSV-1 mutant that expresses the HSV-2 ICP27 instead of the HSV-1 protein. This recombinant was fully sensitive to LMB, indicating that one or more other viral genes must be important in determining HSV-2's LMB-resistant phenotype. In additional work, we report several findings that shed light on how HSV-1 ICP27 mutations can confer LMB resistance. First, we show that LMB treatment of HSV-1-infected cells leads to suppression of late viral protein synthesis and a block to progeny virion release. Second, we identify a novel type of ICP27 mutation that can confer LMB resistance, that being the addition of a 100-residue amino-terminal affinity purification tag. Third, by studying infections where both LMB-sensitive and LMB-resistant forms of ICP27 are present, we show that HSV-1's sensitivity to LMB is dominant to its resistance. Together, our results suggest a model in which the N-terminal portion of ICP27 mediates a nonessential activity that interferes with HSV-1 replication when CRM1 is inactive. We suggest that LMB resistance mutations weaken or abrogate this activity. Herpes simplex virus 1 (HSV-1) is a widely distributed human alphaherpesvirus that is capable of causing serious and in some cases life-threatening infections. The HSV-1 infectious cycle involves both nuclear and cytoplasmic processes, and thus, productive viral replication requires that viral and cellular protein and RNA components travel efficiently between the nucleus and cytoplasm. Such movement is largely mediated by dedicated cellular transporters, one important class of which consists of the karyopherin-␤ family of proteins (1). Among this family, CRM1 is perhaps the most well studied member. It is an essential protein that exports ribosomal subunits and various protein cargos from the nucleus to the cytoplasm (2-4). Transport by CRM1 is dependent on the presence of a specific nuclear export signal (NES) on the cargo protein. This signal consists of an ϳ15-residue consensus sequence that is rich in leucine and other hydrophobic residues. The NES binds to a hydrophobic cleft on the surface of nucleus-localized CRM1 (5, 6). Following binding, CRM1 escorts the cargo through the nuclear pore to the cytoplasm, whereupon it is released. One of the first leucine-rich NESs described was that of the human immunodeficiency virus type 1 (HIV-1) Rev protein (7). Rev uses CRM1 to facilitate the nuclear export of unspliced and partially spliced HIV-1 transcripts. S...

Section: Genetic Basis Of Lmb Resistance In Hsvsupporting

confidence: 51%

Role of Immediate Early Protein ICP27 in the Differential Sensitivity of Herpes Simplex Viruses 1 and 2 to Leptomycin B

Park

Lengyel

Rice

2013

“…Supporting to this hypothesis, more than 15 putative viral and cellular substrates of Us3 have been reported (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). However, among them, only a limited substrates (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), including gB, UL31, Us3 itself, UL47, viral dUTPase (vdUTPase), and tuberous sclerosis complex 2, have been shown both to be physiological Us3 substrates in infected cells and directly linked with Us3 functions in infected cells (11,15,(24)(25)(26)(27)(28).…”

mentioning

confidence: 96%

Phosphorylation of a Herpes Simplex Virus 1 dUTPase by a Viral Protein Kinase, Us3, Dictates Viral Pathogenicity in the Central Nervous System but Not at the Periphery

Shindo

Maruzuru

Kawaguchi

2014

Herpes simplex virus 1 (HSV-1) encodes Us3 protein kinase, which is critical for viral pathogenicity in both mouse peripheral sites (e.g., eyes and vaginas) and in the central nervous systems (CNS) of mice after intracranial and peripheral inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. We recently reported that Us3 phosphorylated HSV-1 dUTPase (vdUTPase) at serine 187 (Ser-187) in infected cells, and this phosphorylation promoted viral replication by regulating optimal enzymatic activity of vdUTPase. In the present study, we show that the replacement of vdUTPase Ser-187 by alanine (S187A) significantly reduced viral replication and virulence in the CNS of mice following intracranial inoculation and that the phosphomimetic substitution at vdUTPase Ser-187 in part restored the wild-type viral replication and virulence. Interestingly, the S187A mutation in vdUTPase had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. Similarly, the enzyme-dead mutation in vdUTPase significantly reduced viral replication and virulence in the CNS of mice after intracranial inoculation, whereas the mutation had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. These observations suggested that vdUTPase was one of the Us3 substrates responsible for Us3 pathogenicity in the CNS and that the CNS-specific virulence of HSV-1 involved strict regulation of vdUTPase activity by Us3 phosphorylation. IMPORTANCEHerpes simplex virus 1 (HSV-1) encodes a viral protein kinase Us3 which is critical for pathogenicity both in peripheral sites and in the central nervous systems (CNS) of mice following peripheral and intracranial inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. Here, we report that Us3 phosphorylation of viral dUTPase (vdUTPase) at serine 187 (Ser-187), which has been shown to promote the vdUTPase activity, appears to be critical for viral virulence in the CNS but not for pathogenic effects in peripheral sites. Since HSV proteins critical for viral virulence in the CNS are, in almost all cases, also involved in viral pathogenicity at peripheral sites, this phosphorylation event is a unique report of a specific mechanism involved in HSV-1 virulence in the CNS.

“…However, to date, although more than 15 putative HSV-1 Us3 substrates have been described (14,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), only a few substrates, including gB, UL31, Us3 itself, UL47, and tuberous sclerosis complex 2, have been shown to be both physiological Us3 substrates in infected cells and directly linked with Us3 functions in infected cells (14,19,20,(22)(23)(24)30). Therefore, there may be Us3 substrates other than those reported to date, and their identification and characterization are required to determine Us3 functions and understand their mechanisms.…”

mentioning

confidence: 99%

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells

Tsuda

Liu

Kozuka‐Hata

et al. 2014