Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage specific E2. Structural, biochemical and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.

BackgroundExpression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration.ResultsThis work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection.ConclusionsThese results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.

dTNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.

Old World monkey TRIM5α proteins are known to block the replication of human immunodeficiency virus and other retroviruses in a species-specific fashion. In this report, we show that specific forms of simian TRIM5α proteins can restrict herpes simplex virus (HSV) infection. To define the effect of TRIM5α on HSV replication, we examined HSV infection in HeLa cell lines that stably express simian and human orthologs of TRIM5α proteins. We demonstrated that several simian TRIM5α proteins can restrict HSV replication, with the TRIM5α protein of rhesus macaques showing the strongest inhibition of HSV infection. We also found that the level of the inhibition of virus replication was viral strain-specific. TRIM5α is likely to inhibit HSV at the early stage of infection; however, at later times of infection, the levels of TRIM5α are significantly decreased. Thus, some TRIM5α proteins exhibit antiviral effects that extend beyond retroviral infections, but HSV may be able to reduce this restriction by reducing TRIM5α levels during the later phases of virus replication. Our results also argue that TRIM5α is only part of the reduced level of HSV replication in rhesus macaques, which are known to be less susceptible to HSV infection than other primates.

The replication-defective herpes simplex virus 2 (HSV-2) dl5-29 mutant virus strain with deletions in the UL5 and UL29 genes has been shown to protect mice and guinea pigs against challenge with wild type (wt) HSV-2 and to protect against ocular disease caused by HSV-1 infection. The dl5-29 strain is currently being prepared for clinical trials as a herpes vaccine candidate. As a possible approach to improve the efficacy of dl5-29 as a genital herpes vaccine, we replaced the UL41 gene encoding the virion host shutoff function (vhs) with the UL41 gene from HSV-1. While the HSV-2 UL41 and HSV-1 UL41 gene products have analogous functions, vhs-1 is 40-fold less active than vhs-2. Previously, it was shown that disruption of the UL41 gene can increase the efficacy of dl5-29 as a vaccine against HSV-2. These properties led us to hypothesize that replacement of vhs-2 by vhs-1 would decrease cytopathic effects in infected host cells, allowing longer survival of antigen-presenting cells and induction of stronger immune responses. The new recombinant dl5-29-41.1 virus shows nearly the same immunogenicity and protection against HSV-2 challenge as the parental dl5-29 virus or a triply deleted mutant virus, dl5-29-41, in the murine model of infection, and grows to higher titers than the parental strain in complementing cells, which is important for GMP production. The results have implications for the design of future HSV-2 vaccine candidates and mechanisms of induction of protective immunity against genital herpes.