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This study was conducted to evaluate the relationship among market weight, slaughter age, yield grade, and primal cut yield in Hanwoo. A total of 403 Hanwoo (Korean native cattle) was assessed for carcass traits such as carcass cold weight, backfat thickness, ribeye area, dressing percentage, yield index, and marbling score. The production yield of the individual major primal cuts of Hanwoo beef was also measured. Carcass cold weight, ribeye area, and backfat thickness, which affect meat quality increased with increased market weight (p < 0.05). The production yield of the ten major primal cuts also increased with increased market weight (p < 0.05). In terms of slaughter age, carcass cold weight, ribeye area, and backfat thickness all increased from 25 months to 28–29 months, and the production yield of all prime cuts also increased with increasing slaughter age. According to the meat yield grade, carcass cold weight and backfat thickness increased from grade A to grade C, although the ribeye area was not affected. The combined findings of the study suggest that slaughtering Hanwoo at the weight of 651–700 kg and 701–750 and age of 28.23 and 29.83 months could be desirable to achieve the best quality and quantity grade of Hanwoo beef. However, the positive correlation of carcass cold weight and backfat thickness, and the negative correlation of the yield index according to primal cuts yield indicated that it is necessary to couple the slaughtering management of cattle with improved genetic and breeding method of Hanwoo to increase the production yield of the major prime cuts of Hanwoo beef.

Viral deubiquitinases (DUBs) regulate cellular innate immunity to benefit viral replication. In human cytomegalovirus (HCMV), the UL48-encoded DUB regulates innate immune responses, including NF-κB signaling. Although UL48 DUB is known to regulate its stability via auto-deubiquitination, its impact on other viral proteins is not well understood. In this study, we investigated the role of UL48 DUB in regulating the ubiquitination of viral proteins by comparing the levels of ubiquitinated viral peptides in cells infected with wild-type virus and DUB active-site mutants using mass spectrometry. We found that ubiquitinated peptides were increased in DUB mutant virus infection for 90% of viral proteins, with the innermost tegument proteins pp150 (encoded by UL32) and pUL48 itself being most significantly affected. The highly deubiquitinated lysine residues of pUL48 were mapped within its N-terminal DUB domain and the nuclear localization signal. Among them, the arginine substitution of lysine 2 (K2R) increased pUL48 stability and enhanced viral growth at low multiplicity of infection, indicating that K2 auto-deubiquitination has a role in regulating pUL48 stability. pUL48 also interacted with pp150 and increased pp150 expression by downregulating its ubiquitination. Furthermore, we found that, unlike the wild-type virus, mutant viruses expressing the UL48 protein with the DUB domain deleted or DUB active site mutated contain higher levels of ubiquitin conjugates, including the ubiquitinated forms of pp150, in their virions. Collectively, our results demonstrate that UL48 DUB mainly acts on the innermost tegument proteins pp150 and pUL48 itself during HCMV infection and may play a role in protecting virions from the inclusion of ubiquitin conjugates. IMPORTANCE Herpesviruses encode highly conserved tegument proteins that contain deubiquitinase (DUB) activity. Although the role of viral DUBs in the regulation of host innate immune responses has been established, their roles in the stability and function of viral proteins are not well understood. In this study, we performed a comparative analysis of the levels of ubiquitinated viral peptides between wild-type and DUB-inactive HCMV infections and demonstrated that the innermost tegument proteins pp150 and pUL48 (DUB itself) are major targets of viral DUB. We also show that ubiquitinated viral proteins are effectively incorporated into the virions of DUB mutant viruses but not the wild-type virus. Our study demonstrates that viral DUBs may play important roles in promoting the stability of viral proteins and inhibiting the inclusion of ubiquitin conjugates into virions.

ObjectiveThis study was conducted to evaluate the relationship between market weight, slaughter age, yield grade, and primal cut yield in Hanwoo. MethodsA total of 403 Hanwoo (Korean native cattle) was assessed for carcass traits such as carcass cold weight, backfat thickness, ribeye area, dressing percentage, yield index, and marbling score. The production yield of the individual major primal cuts of Hanwoo beef was also measured. FindingsCarcass cold weight, ribeye area, and backfat thickness, which affect meat quality increased with increased market weight (p

The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.

Human cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation in trans . Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry. IMPORTANCE HCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

Vaccination is the most efficient method for infectious disease prevention. Parenteral injections such as intramuscular, intradermal, and subcutaneous injections have several advantages in vaccine delivery, but there are many drawbacks. Thus, the development of a new vaccine delivery system has long been required. Recently, microneedles have been attracting attention as new vaccination tools. Microneedle is a highly effective transdermal vaccine delivery method due to its mechanism of action, painlessness, and ease of use. Here, we summarized the characteristics of microneedles and the possibilities as a new vaccine delivery route.

본 연구는 말고기 등급(1등급과 2등급)에 따른 품질을 비교하여 향후 부위별 및 등급별 구분 판매시 등급간 육질 특성의 기초자료로 활용하기 위해 이화학적, 관능적, 지방산조성 분석을 실시하였다. 등심에 대한 일반성분분석 결과에서는 1등급이 2등급에 비해 높은(p $kg/cm^2$ )으로 2등급 10.86 ( $kg/cm^2$ )에 비해 유의적으로 낮았다(p 0.05), 육색에 대한 육질등급별 부위간 비교에서는 $L^*$ 값, $a^*$ 값 및 $b^*$ 값에서 차이가 없었으나, 2등급의 경우 등심, 목심, 넓적다리의 $L^*$ , $a^*$ 및 $b^*$ 값은 유의적 차이가 있는 것으로 나타났다(p 0.05), oleic acid (C18:1)는 등급간에 유의적 차이(p 0.05). 이상의 결과에서 냉동된 말고기의 육질 1등급과 2등급에서는 조지방 함량, 전단력, palmitoleic acid (C16:1)와 stearic acid (C18:0)는 등급별 육질을 판별할 수 있는 요소로 이용이 가능할 것으로 사료된다. 또한 품질을 향상시키고 부위 및 등급에 따른 차별화를 위해서는 40개월 이상의 장기간 사육은 바람직하지 않을 것으로 사료된다. 【The objective of this study was to investigate the physical and chemical composition, fatty acid profile and sensory property of horse meat according to meat quality grade (1 and 2) and cuts (loin, chuck roll and top round). The lipid content of loin was significantly higher (p $kg/cm^2$ ) and 2 (10.86 $kg/cm^2$ ). Water-holding capacity values of loin, chuck roll and top round were not different (p>0.05) between grades 1 and 2. Meat color values ( $L^*$ , $a^*$ and $b^*$ ) of loin, chuck roll and top round were not different (p>0.05) between grades 1 and 2. Palmitoleic acid of loin in grade 1 (11.39%) was higher (p 0.05) between grades 1 and 2. Therefore, meat quality grade had mainly affected lipid and moisture contents, shear force, palmitoleic acid and stearic acid of horse loin; and horse loin of grade 2 had lower pH and higher $L^*$ , $a^*$ and $b^*$ than the other cuts.】

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-γ) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Zβ and D3 domains, but not the Zα domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DAI knockdown did not affect the B-DNA-mediated IFN-β transcription and IRF3 activation, and overexpression of DAI and RIP1 did not enhance NF-κB activation by B-DNA treatment, demonstrating that DAI is not essential for the B-DNA-mediated IFN production in HepG2 cells. DAI colocalized with ICP0 in a subset of nuclear and cytoplasmic foci in infected cells and interacted with ICP0 in coimmunoprecipitation assays. The anti-HSV-1 effect of DAI was not observed in ICP0-deleted mutant virus infection at a high MOI in HepG2 cells and mouse embryonic fibroblasts. Degradation of IFI16 and PML by ICP0 was enhanced in infection of DAI-knockdown cells. Collectively, these results demonstrate that DAI can suppress HSV-1 growth independent of DNA sensing through mechanisms involving suppression of viral genomes and regulation of ICP0.

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection., Author Summary Type I IFN response is a front-line defense against virus infection. Activation of type I IFN signaling leads to expression of a subset of cellular proteins encoded by interferon-stimulated genes (ISGs). ISG15 encodes an ubiquitin-like protein that is covalently conjugated to protein lysine residues. ISG15 modification (ISGylation) of a protein causes changes of protein function. ISGylation is known to inhibit the replication of many viruses, although pro-viral effects of ISGylation are also reported. Given that ISG15 and the enzymes involved in ISGylation are strongly induced upon virus infection, understanding the interplay between virus and ISGylation is an important issue in virus-host interaction. Nevertheless, viral substrates of ISG15 and viral strategies to regulate ISGylation-mediated antiviral responses are limited to only a few examples. In this study we demonstrate that ISGylation suppresses human cytomegalovirus (HCMV) infection but the virus is armed with countermeasures that consecutively reduce ISG15 transcription and protein ISGylation. Interestingly, a viral ISG15 target is found to inhibit ISGylation. This study highlights that ISGylation is a critical innate immune response against HCMV infection and interfering with ISG15-mediated anti-viral immunity is critical for productive viral infection.

Background The A20 ubiquitin-editing enzyme is a target of nuclear factor kappa B (NF-κB) and also plays a key role in regulating the NF-κB signaling pathway. NF-κB activity is increased during human cytomegalovirus (HCMV) infection and HCMV appears to be adapted to this change. To better understand the regulation of NF-κB signaling during HCMV infection, we investigated how A20 expression is controlled during HCMV infection. Methods The expression level of A20 in human fibroblast cells infected with HCMV or UV-inactivated virus (UV-HCMV) was measured by immunoblot analysis, cell staining, and quantitative real-time PCR. Changes of histone modifications on the A20 promoter were determined by chromatin immunoprecipitation assays. Lentiviral vectors were used to knockdown A20 in fibroblast cells. Results A20 expression was increased at early times after HCMV infection. This increase of the A20 protein level was promoted by viral gene expression under low viral load conditions. The viral IE1 protein, which is known to activate NF-κB, increased the A20 promoter activity through the upstream NF-κB sites in reporter assays, suggesting that IE1 is at least partly involved in A20 induction. Analysis of A20 expression with a high viral load demonstrated that the A20 regulation by HCMV was biphasic; both A20 protein and mRNA levels were increased at the early stage of infection, but decreased at the late stage. Under high viral load conditions, A20 upregulation was more profound with UV-HCMV than with HCMV, indicating a role of the viral gene product(s) in limiting A20 induction. Consistently, more histone modifications for euchromatin were found on the A20 promoter during UV-HCMV infection than with HCMV infection. A20 knockdown by shRNA reduced HCMV growth. Conclusion These results suggest that the biphasic regulation of A20 expression may be important for productive HCMV infection.

Deoxyribonucleotides (dNTPs) are important for the efficient growth of DNA viruses. Therefore, many DNA viruses have strategies for the upregulation of cellular dNTP levels. Both α- and γ-herpesviruses encode functional homologs of cellular dNTP anabolic enzymes, including the class I ribonucleotide reductase (RNR) large (R1) and small (R2) subunits, whereas β-herpesviruses modulate host cells to induce genes that increase dNTP levels. Interestingly, β-herpesviruses still express the nonfunctional RNR R1 subunit. However, it is not clear why β-herpesviruses still carry inactive R1 homologs. Recently, the R1 homologs of herpesviruses have been shown to inhibit innate immune signaling pathways. In particular, both functional and nonfunctional R1 homologs target receptor-interacting protein kinase 1 (RIP1) and inhibit RIP1-mediated signaling pathways to promote viral replication. Here, we summarize recent findings on the activity of herpesviral R1 homologs and discuss their roles in the regulation of innate immune signaling pathways.

NF-κB transcription factors are key regulators of immune and stress responses, apoptosis, and differentiation. Human cytomegalovirus (HCMV) activates or represses NF-κB signaling at different times during infection. An initial increase in NF-κB activity occurs within a few hours of infection. The virus appears to adapt to this change since initial viral gene expression is promoted by the elevated NF-κB activity. Because NF-κB upregulates innate immune responses and inflammation, it has also been suggested that HCMV needs to downregulate NF-κB signaling. Recent studies have shown that HCMV has various mechanisms that inhibit NF-κB signaling. HCMV reduces cell surface expression of tumor necrosis factor receptor 1 (TNFR1) and blocks the DNA binding activity of NF-κB. Furthermore, some HCMV tegument proteins antagonize NF-κB activation by targeting the key components of NF-κB signaling at late stages of infection. In this review, we summarize the recent findings on the relationship between HCMV and NF-κB signaling, focusing, in particular, on the viral mechanisms by which the NF-κB signaling pathway is inhibited.

Viruses interact with the host ubiquitination system in a variety of ways. Viral proteins are often a substrate for ubiquitination, which leads to proteasomal degradation. Viruses also have functions to modify the cellular ubiquitination machinery. Recently, deubiquitinating protease (DUB) activity has been found in many viral proteins. In herpesviruses, the DUB domain is found within the large tegument protein, which is conserved in all members of the herpesvirus family. Although a limited number of viral and cellular targets have been identified to date, accumulating evidence shows that herpesviral DUBs may primarily target key cellular regulators of immune signaling pathways to promote viral replication. In this review, we summarize the recent findings on viral DUBs. In particular, we focus on the herpesviral DUBs and their targets, and discuss their potential roles in the regulation of immune signaling pathways.