The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50. IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

IntroductionThe effect of tixagevimab/cilgavimab (Evusheld™; AstraZeneca, UK) should be evaluated in the context of concurrent outbreak situations.MethodsFor serologic investigation of tixagevimab/cilgavimab during the BA.5 outbreak period, sera of immunocompromised (IC) hosts sampled before and one month after tixagevimab/cilgavimab administration and those of healthcare workers (HCWs) sampled one month after a 3rd shot of COVID-19 vaccines, five months after BA.1/BA.2 breakthrough infection (BI), and one month after BA.5 BI were investigated. Semi-quantitative anti-spike protein antibody (Sab) test and plaque reduction neutralizing test (PRNT) against BA.5 were performed.ResultsA total of 19 IC hosts (five received tixagevimab/cilgavimab 300 mg and 14 received 600 mg) and 41 HCWs (21 experienced BA.1/BA.2 BI and 20 experienced BA.5 BI) were evaluated. Baseline characteristics did not differ significantly between IC hosts and HCWs except for age and hypertension. Sab significantly increased after tixagevimab/cilgavimab administration (median 130.2 BAU/mL before tixagevimab/cilgavimab, 5,665.8 BAU/mL after 300 mg, and 10,217 BAU/mL after 600 mg; both P < 0.001). Sab of one month after the 3rd shot (12,144.2 BAU/mL) or five months after BA.1/BA.2 BI (10,455.8 BAU/mL) were comparable with that of tixagevimab/cilgavimab 600 mg, while Sab of one month after BA.5 BI were significantly higher (22,216.0 BAU/mL; P < 0.001). BA.5 PRNT ND50 significantly increased after tixagevimab/cilgavimab administration (median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after 300 mg, and 298.5 after 600 mg; both P < 0.001). The ND50 after tixagevimab/cilgavimab 600 mg was comparable to those of five months after BA.1 BI (ND50 200.9) while ND50 of one month after the 3rd shot was significantly lower (ND50 107.6; P = 0.019). The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among tested groups, but statistical difference was not noticed with tixagevimab/cilgavimab 600 mg.ConclusionTixagevimab/cilgavimab provided a comparable neutralizing activity against the BA.5 with a healthy adult population who were vaccinated with a 3rd shot and experienced BA.1/BA.2 BI.

Sterile alpha motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) acts as a restriction factor for several RNA and DNA viruses by limiting the intracellular pool of deoxynucleoside triphosphates. Here, we investigated the regulation of SAMHD1 expression during human cytomegalovirus (HCMV) infection. SAMHD1 knockdown using shRNA increased the activity of the viral UL99 late gene promoter in human fibroblasts by 7-to 9-fold, confirming its anti-HCMV activity. We also found that the level of SAMHD1 was initially increased by HCMV infection but decreased partly at the protein level at late stages of infection. SAMHD1 loss was not observed with UV-inactivated virus and required viral DNA replication. This reduction of SAMHD1 was effectively blocked by MLN4924, an inhibitor of the Cullin-RING-E3 ligase (CRL) complexes, but not by bafilomycin A1, an inhibitor of vacuolar-type H +-ATPase. Indirect immunofluorescence assays further supported the CRL-mediated SAMHD1 loss at late stages of virus infection. Knockdown of CUL2 and to a lesser extent CUL1 using siRNA stabilized SAMHD1 in normal fibroblasts and inhibited SAMHD1 loss during virus infection. Altogether, our results demonstrate that SAMHD1 inhibits the growth of HCMV, but HCMV causes degradation of SAMHD1 at late stages of viral infection through the CRL complexes.

Tixagevimab/cilgavimab is a monoclonal antibody used to prevent coronavirus disease 2019 among immunocompromised hosts and maintained neutralizing activity against early omicron variants. Omicron BN.1 became a dominant circulating strain in Korea early 2023, but its susceptibility to tixagevimab/cilgavimab is unclear. We conducted plaque reduction neutralization test (PRNT) against BN.1 in a prospective cohort (14 patients and 30 specimens). BN.1 PRNT was conducted for one- and three-months after tixagevimab/cilgavimab administration and the average PRNT ND 50 of each point was lower than the positive cut-off value of 20 (12.9 ± 4.5 and 13.2 ± 4.2, respectively, P = 0.825). In the paired analyses, tixagevimab/cilgavimab-administered sera could not actively neutralize BN.1 (PRNT ND 50 11.5 ± 2.9, P = 0.001), compared with the reserved activity against BA.5 (ND 50 310.5 ± 180.4). Unlike virus-like particle assay, tixagevimab/cilgavimab was not active against BN.1 in neutralizing assay, and would not be effective in the present predominance of BA.2.75 sublineages.

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 wild-type and DUB active-site mutant virus-infected cells 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 or 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 demonstrate 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.