Kinase Us3 Research Articles

Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus.

The alphaherpesvirus UL34 protein is necessary for the primary envelopment of intranuclear capsids at the inner leaflet of the nuclear membrane. In herpes simplex virus type 1, the UL34 protein is exclusively phosphorylated by the protein kinase encoded by the non-essential US3 gene. To investigate the effect of the pseudorabies virus (PrV) US3 product on the intracellular localization of the UL34 protein and on virus morphogenesis, PrV US3 deletion mutants were isolated and characterized. Immunofluorescence analyses demonstrated that in the absence of the US3 protein, the localization of the UL34 polypeptide to the nuclear membrane was not as pronounced as that seen with US3, although immunoelectron microscopy indicated the presence of the UL34 protein in both leaflets of the nuclear membrane. Ultrastructurally, an accumulation of enveloped virions in the perinuclear space in large invaginations of the inner nuclear membrane was observed, which were shown by immunoelectron microscopy to contain the UL34 protein, but not glycoproteins gB or gC. Thus, the US3 protein appears to be involved in the de-envelopment of perinuclear virions by fusion with the outer leaflet of the nuclear membrane. Surprisingly, no difference in the phosphorylation of the PrV UL34 protein was observed in the presence or absence of the US3 kinase. Therefore, the observed effects of the PrV US3 protein on the intracellular localization of the UL34 protein and on virus morphogenesis are probably not due to the phosphorylation of the UL34 protein by the US3 kinase.

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids.

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

The US3 protein kinase of herpes simplex virus 1 mediates the posttranslational modification of BAD and prevents BAD-induced programmed cell death in the absence of other viral proteins.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome c, activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U(S)3 delivered in trans blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U(S)3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U(S)3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U(S)3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.

The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus.

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (alpha4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled US3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type alpha4 gene but from which the US3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the US3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the US3 gene as the principal viral product required to block apoptosis.

The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation of the regulatory protein alpha 22.

The herpes simplex virus 1 genome was shown to encode two genes, US3 and UL13, exhibiting amino acid sequence motifs common to protein kinases. Elsewhere this laboratory reported that the prominent substrate of the US3 protein kinase is the product of the UL34 gene, an essential nonglycosylated membrane protein. In the absence of the US3 kinase, the UL34 protein remains unphosphorylated but forms a complex with four proteins that become phosphorylated uniquely when UL34 is not. To investigate the role of UL13 protein in this process, recombinant viruses lacking UL13 or both UL13 and US3 were constructed. We report that UL13 is dispensable for viral replication in cell culture and is not involved in the processing of UL34 or of associated phosphoproteins. UL13 is, however, responsible for the posttranslational processing associated with phosphorylation of infected-cell protein 22, the product of the alpha 22 gene. This gene was previously reported to play a regulatory role in selected cell lines. UL13 appears to be either a protein kinase or a phosphotransferase and its major substrate is the alpha 22 protein.