The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4 DCAF1 ). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1-and CUL4-associated factor 1 (DCAF1 The viral protein R (Vpr) 2 is one of four HIV-1 accessory proteins (Nef, Vif, Vpr, and Vpu), that regulate virus infectivity, primarily through interactions with host proteins (1). Vpr is highly conserved in HIV-1, HIV-2, and the simian immunodeficiency viruses (2-4). Several biological roles for Vpr during viral infection of cells have been described, including facilitation of nuclear translocation of preintegration complexes (5-7), modulation of mutation frequency (8, 9), induction of cell cycle arrest in the G 2 /M phase (10 -15), and stimulation of host cell apoptosis (16 -18). More recently, Vpr has been postulated to enhance infection by mediating the degradation of unknown cellular defense factors (1, 19).To date, three of the four HIV-1 accessory proteins, including Vpr, have been found to interact with cullin-RING finger E3 ubiquitin ligases (CRLs). E3 ligases are multisubunit complexes that include a cullin (CUL), a RING H2 finger protein (RBX1), an adaptor, and a substrate recognition subunit (20). More specifically, Vpr interacts with the CRL4 DCAF1 E3 ubiquitin ligase, assembled with cullin 4A (CUL4A), RBX1, DDB1 (DNA damage-binding protein 1), and DCAF1 (DDB1-and CUL4-associated factor 1) (1, 21). The substrate recognition subunit of this CRL4, DCAF1, previously known as Vpr-binding protein (VprBP), was originally identified via co-precipitation with Vpr (22). At the present time, substantial evidence suggests that Vpr usurps CRL4 DCAF1 E3 ubiquitin ligases to ubiquitinate (ubiquitylate) and degrade unknown cellular proteins required for cell cycle progression (23-31). In fact, Vpr was reported to bind and modulate the activity of cell cycle-related proteins, such as CDC25 (32), WEE1 kinase (33), and SAP145 (34, 35). In addition, Vpr activates ATM and Rad3-related checkpoint kinase (ATR)-dependent DNA damage signaling pathways, including phosphorylation of the histone 2A variant-X and BRCA1 (36, 37). However, whether Vpr interactions with these proposed host cellular factors result in their degradation via CRL4 DCAF1-Vpr E3 ubiquitin ligases had not been established.One potential target of Vpr is uracil-DNA glycosylase-2 (UNG2), which removes uracil lesions from single-stranded and double-stranded DNA in the base excision repair pathway. Initially identified in a yeast two-hybrid screen (38), UNG2 has been implicated as a Vpr-dependent substrate of the CRL4 E3 ubiquitin ligase (39,40).However, a number of studies investigating the roles of UNG2 in HIV replication resulted in opposing views regarding HIV biology. For example, some studies found that virion-associated UNG2 modulates the innate ...

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction.

Introduction Family presence during emergency resuscitations is increasingly common, but the question remains whether the practice results in psychological harm to the witness. We examine whether family members who witness resuscitations have increased post-traumatic stress disorder (PTSD) symptoms at one month following the event. Methods We identified family members of critically ill patients via our emergency department (ED) electronic health record. Patients were selected based on their geographic triage to an ED critical care room. Family members were called a median of one month post-event and administered the Impact of Event Scale-Revised (IES-R), a 22-item validated scale that measures post-traumatic distress symptoms and correlates closely with Diagnostic and Statistical Manual of Mental Disorders-IV criteria for post-traumatic stress disorder (PTSD). Family members were placed into two groups based on whether they stated they had witnessed the resuscitation (FWR group) or not witnessed the resuscitation (FNWR group). Data analyses included chi-square test, independent sample t-test, and linear regression controlling for gender and age. Results A convenience sample of 423 family members responded to the phone interview: 250 FWR and 173 FNWR. The FWR group had significantly higher mean total IES-R scores: 30.4 vs 25.6 (95% confidence interval [CI], −8.73 to −0.75; P<.05). Additionally, the FWR group had significantly higher mean score for the subscales of avoidance (10.6 vs 8.1; 95% CI, −4.25 to −0.94; P<.005) and a trend toward higher score for the subscale of intrusion (13.0 vs 11.4; 95% CI, −3.38 to .028; P = .054). No statistical significant difference was noted between the groups in the subscale of hyperarousal (6.95 vs 6.02; 95% CI, −2.08 to 0.22; P=.121). All findings were consistent after controlling for age, gender, and immediate family member (spouse, parent, children, and grandchildren). Conclusion Our results suggest that family members who witness ED resuscitations may be at increased risk of PTSD symptoms at one month. This is the first study that examines the effects of family visitation for an unsorted population of very sick patients who would typically be seen in the critical care section of a busy ED.

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