Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1

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To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication. IMPORTANCEHerpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment. Herpesviruses have a unique virion structure designated the tegument, which is a proteinaceous layer consisting of a number of different viral proteins and is located between the nucleocapsid and the envelope (1). Herpes simplex virus 1 (HSV-1), classified in the Alphaherpesvirinae subfamily of the Herpesviridae family, is one of the best-studied members in the family and has at least 23 different viral proteins in the tegument (2).In HSV-1-infected cells, packaging of nascent progeny...

Section: Cells and Virusesmentioning

confidence: 99%

The Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis

Oda

Arii

Koyanagi

et al. 2016

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“…Vero, HEp-2, and rabbit skin cells were described previously (27,28). The following viruses have been described previously (21,29): HSV-1 wild-type strain HSV-1(F); recombinant virus YK524, encoding UL47 fused to the monomeric red fluorescent protein mRFP1 (mRFP1-UL47); recombinant virus YK527, encoding mRFP1-UL47 and carrying the Us3K220M mutation (mRFP1-UL47/Us3K220M); recombinant virus YK528, in which Us3K220M in YK527 was repaired (mRFP1-UL47/Us3K220M-repair); recombinant virus YK523, encoding mRFP1-UL47 and Us3 fused to the fluorescent protein VenusA206K (VenusA206K-Us3/mRFP1-UL47); recombinant virus YK545, a UL47-null mutant virus in which the UL47 gene was disrupted by insertion of a foreign gene cassette just downstream of the UL47 start codon (⌬UL47); and recombinant virus YK546, in which the foreign gene cassette inserted into the UL47 locus of YK545 (⌬UL47) was excised (⌬UL47-repair) (Fig.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…To generate rabbit polyclonal antibody to UL31 or UL47, rabbits were immunized, respectively, with purified MBP-UL31(2)-Pii or with a mixture of MBP-UL47-Pi, MBP-UL47-Pii, and MBP-UL47-Piii as described previously (27). Serum from the immunized rabbits was used as anti-UL31 or anti-UL47 rabbit polyclonal antibody.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Herpes Simplex Virus 1 UL47 Interacts with Viral Nuclear Egress Factors UL31, UL34, and Us3 and Regulates Viral Nuclear Egress

Liu

Kato

Shindo

et al. 2014

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Herpesviruses have evolved a unique mechanism for nuclear egress of nascent progeny nucleocapsids: the nucleocapsids bud through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes (primary envelopment), and enveloped nucleocapsids then fuse with the outer nuclear membrane to release nucleocapsids into the cytoplasm (de-envelopment). We have shown that the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (or VP13/VP14) is a novel regulator for HSV-1 nuclear egress. In particular, we demonstrated the following: (i) UL47 formed a complex(es) with HSV-1 proteins UL34, UL31, and/or Us3, which have all been reported to be critical for viral nuclear egress, and these viral proteins colocalized at the nuclear membrane in HSV-1-infected cells; (ii) the UL47-null mutation considerably reduced primary enveloped virions in the perinuclear space although capsids accumulated in the nucleus; and (iii) UL47 was detected in primary enveloped virions in the perinuclear space by immunoelectron microscopy. These results suggested that UL47 promoted HSV-1 primary envelopment, probably by interacting with the critical HSV-1 regulators for viral nuclear egress and by modulating their functions. IMPORTANCELike other herpesviruses, herpes simplex virus 1 (HSV-1) has evolved a vesicle-mediated nucleocytoplasmic transport mechanism for nuclear egress of nascent progeny nucleocapsids. Although previous reports identified and characterized several HSV-1 and cellular proteins involved in viral nuclear egress, complete details of HSV-1 nuclear egress remain to be elucidated. In this study, we have presented data suggesting (i) that the major HSV-1 virion structural protein UL47 (or VP13/VP14) formed a complex with known viral regulatory proteins critical for viral nuclear egress and (ii) that UL47 played a regulatory role in HSV-1 primary envelopment. Thus, we identified UL47 as a novel regulator for HSV-1 nuclear egress.M orphogenesis of herpes simplex virus 1 (HSV-1), like that of other herpesviruses, takes place in two different cellular compartments (1, 2). Viral DNA replication and transcription, capsid assembly, and packaging of nascent progeny virus genomes into preformed capsids take place in the nucleus, and final envelopment takes place in the cytoplasm (1, 2). Since herpesvirus nucleocapsids are too large to traverse the nuclear lamina or cross the inner and outer nuclear membranes (INM and ONM, respectively) through nuclear pores, these viruses appear to have evolved a unique nuclear egress mechanism in which progeny nucleocapsids acquire primary envelopes by budding through the INM into the space between the INM and ONM, the perinuclear space, and then the enveloped nucleocapsids fuse with the ONM to release de-enveloped nucleocapsids into the cytoplasm (1, 2).In the present study, we focus on the first step of HSV-1 nuclear egress, the process by which progeny nucleocapsids acquire primary envelopes by budding through the INM into the perinuclear space (...

“…Vero, rabbit skin, HEK293T, Plat-GP, and HEp-2 cells were described previously (32)(33)(34)(35). sh-Luc-HEp-2 cells expressing short hairpin RNA (shRNA) against firefly luciferase were described previously (36).…”

Section: Cells and Virusesmentioning

confidence: 99%

Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication

Sato

Maruzuru

Oyama

et al. 2016

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To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication. IMPORTANCEUpon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulates viral chromatin remodeling for efficient viral gene expression and replication. H erpes simplex virus 1 (HSV-1) has more than 80 different genes that fall into three major classes, designated immediate early (IE) or ␣, early (E) or ␤, and late (L) or ␥, which are expressed in a regulated cascade during the HSV-1 lytic infection cycle (1). ICP0, the subject of this study, is an IE protein with a RING finger domain that confers E3 ubiquitin ligase activity, thereby mediating the ubiquitination and proteasome-dependent degradation of target proteins in HSV-1-infected cells (1-4). Based on studies using ICP0-null mutant viruses, ICP0 has been shown to be required for efficient HSV-1 gene expression and replication in cell cultures (3-7). Numerous studies of ICP0 have gradually identified the mechanisms by which ICP0 acts in HSV-1-infected cells as follows. (i) ICP0 induces the disruption of nuclear structures, designated ND10, by degrading promyelocytic leukemia protein (PML) and Sp100, major cellula...