Herpes simplex virus type 2 tegument protein UL56 relocalizes ubiquitin ligase Nedd4 and has a role in transport and/or release of virions

<p>Abstract</p> <p>Background</p> <p>The ubiquitin system functions in a variety of cellular processes including protein turnover, protein sorting and trafficking. Many viruses exploit the cellular ubiquitin system to facilitate viral replication. In fact, herpes simplex virus (HSV) encodes a ubiquitin ligase (E3) and a de-ubiquitinating enzyme to modify the host's ubiquitin system. We have previously reported HSV type 2 (HSV-2) tegument protein UL56 as a putative adaptor protein of neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) E3 ligase, which has been shown to be involved in protein sorting and trafficking.</p> <p>Results</p> <p>In this study, we visualized and characterized the dynamic intracellular localization of UL56 and Nedd4 using live-cell imaging and immunofluorescence analysis. UL56 was distributed to cytoplasmic vesicles, primarily to the trans-Golgi network (TGN), and trafficked actively throughout the cytoplasm. Moreover, UL56 relocalized Nedd4 to the vesicles in cells transiently expressing UL56 and in cells infected with HSV-2. We also investigated whether UL56 influenced the efficiency of viral replication, and found that extracellular infectious viruses were reduced in the absence of UL56.</p> <p>Conclusion</p> <p>These data suggest that UL56 regulates Nedd4 and functions to facilitate the cytoplasmic transport of virions from TGN to the plasma membrane and/or release of virions from the cell surface.</p

Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

<p>Abstract</p> <p>Background</p> <p>Herpes simplex viruses (HSVs) rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1), a host cell protein markedly up-regulated by HSV infection.</p> <p>Results</p> <p>INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP) assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA.</p> <p>Conclusions</p> <p>The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.</p

Herpes simplex virus type 1 UL14 tegument protein regulates intracellular compartmentalization of major tegument protein VP16

<p>Abstract</p> <p>Background</p> <p>Herpes simplex virus type 1 (HSV-1) has a complicated life-cycle, and its genome encodes many components that can modify the cellular environment to facilitate efficient viral replication. The protein UL14 is likely involved in viral maturation and egress (Cunningham C. et al), and it facilitates the nuclear translocation of viral capsids and the tegument protein VP16 during the immediate-early phase of infection (Yamauchi Y. et al, 2008). UL14 of herpes simplex virus type 2 exhibits multiple functions (Yamauchi Y. et al, 2001, 2002, 2003).</p> <p>Methods</p> <p>To better understand the function(s) of UL14, we generated VP16-GFP-incorporated UL14-mutant viruses with either single (K51M) or triple (R60A, R64A, E68D) amino acid substitutions in the heat shock protein (HSP)-like sequence of UL14. We observed the morphology of cells infected with UL14-null virus and amino acid-substituted UL14-mutant viruses at different time points after infection.</p> <p>Results</p> <p>UL14(3P)-VP16GFP and UL14D-VP16GFP (UL14-null) viruses caused similar defects with respect to growth kinetics, compartmentalization of tegument proteins, and cellular morphology in the late phase. Both the UL14D-VP16GFP and UL14(3P)-VP16GFP viruses led to the formation of an aggresome that incorporated some tegument proteins but did not include nuclear-egressed viral capsids.</p> <p>Conclusions</p> <p>Our findings suggest that a cluster of charged residues within the HSP-like sequence of UL14 is important for the molecular chaperone-like functions of UL14, and this activity is required for the acquisition of functionality of VP16 and UL46. In addition, UL14 likely contributes to maintaining cellular homeostasis following infection, including cytoskeletal organization. However, direct interactions between UL14 and VP16, UL46, or other cellular or viral proteins remain unclear.</p

Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

<p>Abstract</p> <p>Background</p> <p>Herpes simplex virus type 2 (HSV-2) is one of many viruses that exploits and modifies the cellular ubiquitin system. HSV-2 expresses the tegument protein UL56 that has been implicated in cytoplasmic transport and/or release of virions, and is a putative regulatory protein of Nedd4 ubiquitin ligase. In order to elucidate the biological function of UL56, this study examined the interaction of UL56 with the Nedd4-family ubiquitin ligase Itch and its role in the regulation of Itch. Additionally, we assessed the similarity between UL56 and regulatory proteins of Itch and Nedd4, Nedd4-family-interactins proteins (Ndfip).</p> <p>Results</p> <p>UL56 interacted with Itch, independent of additional viral proteins, and mediated more striking degradation of Itch, compared to Nedd4. Moreover, it was suggested that the lysosome pathway as well as the proteasome pathway was involved in the degradation of Itch. Other HSV-2 proteins with PY motifs, such as VP5 and VP16, did not mediate the degradation of endogenous Itch. Ndfip1 and Ndfip2 were similar in subcellular distribution patterns to UL56 and colocalized with UL56 in co-transfected cells.</p> <p>Conclusions</p> <p>We believe that this is the first report demonstrating the interaction of a HSV-specific protein and Itch. Thus, UL56 could function as a regulatory protein of Itch. The mechanism, function and significance of regulating Itch in HSV-2 infection remain unclear and warrant further investigation.</p

Immunization with a highly attenuated replication-competent herpes simplex virus type 1 mutant, HF10, protects mice from genital disease caused by herpes simplex virus type 2

Genital herpes is an intractable disease caused mainly by herpes simplex virus (HSV) type 2 (HSV-2), and is a major concern in public health. A previous infection with HSV type 1 (HSV-1) enhances protection against primary HSV-2 infection to some extent. In this study, we evaluated the ability of HF10, a naturally occurring replication-competent HSV-1 mutant, to protect against genital infection in mice caused by HSV-2. Subcutaneous inoculation of HF10-immunized mice against lethal infection by HSV-2, and attenuated the development of genital ulcer diseases. Immunization with HF10 inhibited HSV-2 replication in the mouse vagina, reduced local inflammation, controlled emergence of neurological dysfunctions of HSV-2 infection, and increased survival. In HF10-immunized mice, we observed rapid and increased production of interferon-γ in the vagina in response to HSV-2 infection, and numerous CD4+ and a few CD8+ T cells localized to the infective focus. CD4+ T cells invaded the mucosal subepithelial lamina propria. Thus, the protective effect of HF10 was related to induction of cellular immunity, mediated primarily by Th1 CD4+ cells. These data indicate that the live attenuated HSV-1 mutant strain HF10 is a promising candidate antigen for a vaccine against genital herpes caused by HSV-2

Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection

Spatiotemporal T790M Heterogeneity in Individual Patients with EGFR-Mutant Non–Small-Cell Lung Cancer after Acquired Resistance to EGFR-TKI

IntroductionEpidermal growth factor receptor (EGFR) mutation T790M accounts for approximately half of acquired resistances to EGFR-tyrosine kinase inhibitor (TKI). Because T790M is mediated by TKI exposure, its penetration and “on–off” may affect T790M status.MethodsWe retrospectively reviewed T790M status and clinical course of patients who had undergone multiple rebiopsies after acquired resistance to EGFR-TKI.ResultsOf 145 patients with EGFR-mutant NSCLC receiving rebiopsy after acquired resistance, 30 underwent multiple site rebiopsies, and 24 received repeated rebiopsies at the same lesion. In 22 patients who underwent rebiopsies from both central nervous system (CNS; 20 cerebrospinal fluids [CSF] and 2 brain tumoral tissues) and thoracic lesions (7 lung tissues, 14 pleural effusions, and 1 lymph node), 12 were thoracic-T790M-positive. Of these 12 patients, 10 were CNS-T790M-negative, despite exhibiting thoracic-T790M-positive. All 10 thoracic-T790M-negatives were CNS-T790M-negative. Three patients revealed a spatial heterogeneous T790M status among their thoracic lesions. In 24 patients receiving repeated rebiopsies at the same lesion (12 lung tissues, 6 CSFs, and 6 pleural effusions), T790M status of lung lesions varied in five patients after TKI-free interval. In all five patients whose T790M status changed from positive to negative, EGFR-TKI rechallenge was effective. In three of these five patients, after further TKI exposure, T790M status changed from negative to positive again. There was also a patient whose CSF T790M status changed from negative to positive after high-dose erlotinib therapy.ConclusionsT790M status in an individual patient can be spatiotemporally heterogeneous because of selective pressure from EGFR-TKI

How Sensitive Are Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors for Squamous Cell Carcinoma of the Lung Harboring EGFR Gene–Sensitive Mutations?

Introduction:Epidermal growth factor receptor (EGFR) mutations are found mostly in adenocarcinoma, and rarely in squamous cell carcinoma (SQC). Little is known about SQC harboring EGFR mutations.Methods:Between April 2006 and October 2010, we investigated the incidence of EGFR activating mutations in SQC of the lung using the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method. The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) was retrospectively evaluated in patients with EGFR-mutated SQC. Further pathologic analyses were performed using immunohistochemistry.Results:Thirty-three of 249 patients with SQC (13.3%) had EGFR mutations, including exon 19 deletion (19 of 33 patients, 58%), L858R point mutation in exon 21 (12 of 33, 36%), and G719S point mutation in exon 18 (2 of 33, 6%). Twenty of these 33 patients received EGFR-TKI therapy, and five of these 20 responded to EGFR-TKIs with a response rate of 25.0% (95% confidence interval [CI], 8.7%–49.1%). The patients’ median progression-free survival and median overall survival were 1.4 months (95% CI, 0.7–5.8 months) and 14.6 months (95% CI, 2.9–undeterminable months), respectively. Approximately one third of the EGFR-mutated SQC patients achieved progression-free survival for longer than 6 months. Some of these patients had high carcinoembryonic antigen levels or a history of never smoking, or were positive for thyroid transcription factor-1.Conclusions:Although EGFR-TKIs seem to be generally less effective in EGFR-mutated SQC than in EGFR-mutated adenocarcinoma, some EGFR-mutated SQC patients can obtain clinical benefit from EGFR-TKIs. To better identify these patients, not only EGFR mutation status, but also clinical factors and pathologic findings should be taken into consideration

Infectious virus shedding duration reflects secretory IgA antibody response latency after SARS-CoV-2 infection

新型コロナウイルス排出と粘膜抗体の関係を解明 --呼吸器ウイルスのヒト間伝播を制御・予防する第一歩--. 京都大学プレスリリース. 2023-12-25.Articles: Infectious virus shedding duration reflects secretory IgA antibody response latency after SARS-CoV-2 infection. 京都大学プレスリリース. 2023-12-25.Infectious virus shedding from individuals infected with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is used to estimate human-to-human transmission risk. Control of SARS-CoV-2 transmission requires identifying the immune correlates that protect infectious virus shedding. Mucosal immunity prevents infection by SARS-CoV-2, which replicates in the respiratory epithelium and spreads rapidly to other hosts. However, whether mucosal immunity prevents the shedding of the infectious virus in SARS-CoV-2-infected individuals is unknown. We examined the relationship between viral RNA shedding dynamics, duration of infectious virus shedding, and mucosal antibody responses during SARS-CoV-2 infection. Anti-spike secretory IgA antibodies (S-IgA) reduced viral RNA load and infectivity more than anti-spike IgG/IgA antibodies in infected nasopharyngeal samples. Compared with the IgG/IgA response, the anti-spike S-IgA post-infection responses affected the viral RNA shedding dynamics and predicted the duration of infectious virus shedding regardless of the immune history. These findings highlight the importance of anti-spike S-IgA responses in individuals infected with SARS-CoV-2 for preventing infectious virus shedding and SARS-CoV-2 transmission. Developing medical countermeasures to shorten S-IgA response time may help control human-to-human transmission of SARS-CoV-2 infection and prevent future respiratory virus pandemics