scholarly journals Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane

2021 ◽  
Vol 17 (12) ◽  
pp. e1010132
Author(s):  
Vesa Aho ◽  
Sami Salminen ◽  
Salla Mattola ◽  
Alka Gupta ◽  
Felix Flomm ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Nuclear Export ◽  
Single Particle Tracking ◽  
Structural Reorganization ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Intranuclear Transport ◽  
Simplex Virus

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.

scholarly journals Differentiating the Roles of UL16, UL21, and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Jie Gao ◽  
Renée L. Finnen ◽  
Maxwell R. Sherry ◽  
Valerie Le Sage ◽  
Bruce W. Banfield
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Viral Proteins ◽  
Virus Infections ◽  
Antiviral Therapies ◽  
Nuclear Egress ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2 ◽  
In Cells

ABSTRACT Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress. IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

scholarly journals Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17+)

2007 ◽  
Vol 82 (6) ◽  
pp. 3109-3124 ◽  
Author(s):  
Claus-Henning Nagel ◽  
Katinka Döhner ◽  
Mojgan Fathollahy ◽  
Tanja Strive ◽  
Eva Maria Borst ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Cre Recombinase ◽  
Nuclear Pore ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cytoplasmic Membranes ◽  
Simplex Virus

ABSTRACT To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17+)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17+)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17+)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.

scholarly journals Differentiating the Roles of UL16, UL21 and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids

2020 ◽  
Author(s):  
Jie Gao ◽  
Renée L. Finnen ◽  
Maxwell R. Sherry ◽  
Valerie Le Sage ◽  
Bruce W. Banfield
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Virus Infections ◽  
Viral Capsids ◽  
Nuclear Egress ◽  
Transmission Electron ◽  
Simplex Virus ◽  
In Cells

AbstractPrevious studies from our laboratory established that pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex type 2 (HSV-2) capsids. To better understand the role of these proteins in nuclear egress, we wished to establish whether nuclear egress complex (NEC) localization and/or function was altered in the absence of either pUL16 or pUL21. We used antiserum raised against HSV-2 NEC components pUL31 and pUL34 to examine NEC localization by immunofluorescence microscopy. NEC localization in cells infected with pUL16 deficient viruses was indistinguishable from that observed in cells infected with wild type viruses. By contrast, NEC localization was found to be aberrant in cells infected with pUL21 deficient virus and, instead, showed some similarity to the aberrant NEC localization pattern observed in cells infected with pUs3 deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher resolution examination of nuclear envelope ultrastructure in cells infected with pUL21 deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21 deficient virus infections was found to be dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16 or both pUs3 and pUL16 were consistent with a role for pUL16 upstream of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.Author summaryThe membrane deformation activity of the herpesvirus nuclear egress complex (NEC), allows viral capsids to transit from their site of assembly in the nucleus through both nuclear membranes into the cytoplasm. The timing, extent and directionality of NEC activity must be precisely controlled during viral infection, yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus type 2 (HSV-2) capsids, function to promote nuclear egress, we examined how the lack of each protein impacted NEC localization. These analyses revealed a function of pUL16 in nuclear egress that is distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of viral capsids is a common feature of the replicative cycle of all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process that could be applied to the development of herpes-specific antiviral drugs.

scholarly journals Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Fumio Maeda ◽  
Jun Arii ◽  
Yoshitaka Hirohata ◽  
Yuhei Maruzuru ◽  
Naoto Koyanagi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Null Mutation ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM. IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

scholarly journals Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

2009 ◽  
Vol 84 (4) ◽  
pp. 2110-2121 ◽  
Author(s):  
Ken Sagou ◽  
Masashi Uema ◽  
Yasushi Kawaguchi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

scholarly journals Association of the Herpes Simplex Virus Type 1 Us11 Gene Product with the Cellular Kinesin Light-Chain-Related Protein PAT1 Results in the Redistribution of Both Polypeptides

2003 ◽  
Vol 77 (17) ◽  
pp. 9192-9203 ◽  
Author(s):  
Louisa Benboudjema ◽  
Matthew Mulvey ◽  
Yuehua Gao ◽  
Sanjay W. Pimplikar ◽  
Ian Mohr
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Light Chain ◽  
Herpes Simplex Virus Type ◽  
Nuclear Export ◽  
Kinesin Light Chain ◽  
Dsrna Binding ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components.

Identification of Nuclear Export Signal in UL37 Protein of Herpes Simplex Virus Type 2

2000 ◽  
Vol 276 (3) ◽  
pp. 1248-1254 ◽  
Author(s):  
Daisuke Watanabe ◽  
Yoko Ushijima ◽  
Fumi Goshima ◽  
Hiroki Takakuwa ◽  
Yasushi Tomita ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Simplex Virus ◽  
Export Signal

scholarly journals Processing of α-Globin and ICP0 mRNA in Cells Infected with Herpes Simplex Virus Type 1 ICP27 Mutants

2000 ◽  
Vol 74 (16) ◽  
pp. 7307-7319 ◽  
Author(s):  
Kimberly S. Ellison ◽  
Stephen A. Rice ◽  
Robert Verity ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Export ◽  
Rna Splicing ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Nuclear Retention ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of viral gene expression that modulates RNA splicing, polyadenylation, and nuclear export. We have previously reported that ICP27 causes the cytoplasmic accumulation of unspliced α-globin pre-mRNA. Here we examined the effects of a series of ICP27 mutations that alter important functional regions of the protein on the processing and nuclear transport of α-globin and HSV ICP0 RNA. The results demonstrate that ICP27 mutants that are impaired for growth in noncomplementing cells, including mutants in the N- and C-terminal regions, are defective in the accumulation of α-globin pre-mRNA. Unexpectedly, several mutants that are competent to repress the expression of reporter genes in transient transfection assays failed to accumulate unspliced RNA, implying that different mechanisms are responsible for transrepression and pre-mRNA accumulation. Several mutants caused a marked increase in the length and heterogeneity of the α-globin mRNA poly(A) tail, suggesting that ICP27 may directly or indirectly affect the regulation of poly(A) polymerase. ICP27 was also required for the accumulation of multiple ICP0 intron-bearing transcripts, but this effect displayed a mutational sensitivity profile different from that of accumulation of unspliced α-globin RNA. Moreover, unlike spliced and unspliced α-globin RNAs, which were efficiently exported to the cytoplasm, spliced and intron-containing ICP0 transcripts were predominantly nuclear in localization, and ICP27 was not required for nuclear retention of the spliced message. We propose that these transcript- and ICP27 allele-specific differences may be explained by the presence of a strong cis-acting ICP27 response element in the α-globin transcript.

scholarly journals Inhibition of the Host Translation Shutoff Response by Herpes Simplex Virus 1 Triggers Nuclear Envelope-Derived Autophagy

2013 ◽  
Vol 87 (7) ◽  
pp. 3990-3997 ◽  
Author(s):  
K. Radtke ◽  
L. English ◽  
C. Rondeau ◽  
D. Leib ◽  
R. Lippe ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus
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