Biomedical subjects

N P Klein

Publications and source records attributed to N P Klein.

4 recordsLinked to original sources

PubMed1999

Src kinases involved in hepatitis B virus replication.

Chronic infection by hepatitis B virus is a leading cause of human liver cancer and liver disease. The hepatitis B virus HBx protein is a regulatory factor that is essential for virus infection in mammals and is implicated in development of liver cancer and liver disease. Among the reported activities of HBx is the ability to stimulate Src tyrosine kinases, Ras-GTPases and transcriptional activation. We now demonstrate that HBx activation of Src tyrosine kinases, but not Ras, promotes a high level of viral replication in cell culture. HBx is shown to stimulate reverse transcription of the viral pregenomic mRNA into genomic DNA through a Src-mediated pathway in tissue culture cells. Targeted inhibition of Src tyrosine kinase activity, mutational inactivation of the HBx gene or retargeting of HBx to the nucleus to abolish cytoplasmic signal transduction activity, are shown to impair viral reverse transcription strongly. These studies implicate HBx stimulation of the Src family of tyrosine kinases in stimulation of viral polymerase activity.

Base Sequence↗

PubMed1997

Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras.

The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.

3T3 Cells↗

PubMed1992

trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes.

The human immunodeficiency virus type 1 Rev protein controls expression of certain viral RNAs by binding to these RNAs in the nucleus. To investigate how dominant negative Rev mutants inhibit Rev function, we fused such mutants to hormone-dependent localization signals from the glucocorticoid receptor. Each was found to have fully potent inhibitory activity whether expressed in the nucleus or in the cytoplasm. Wild-type Rev colocalized with an inhibitory fusion protein, implying that the two proteins interact. The resulting complexes accumulated within nuclei in response to steroids but had no effect on expression of Rev-responsive mRNAs. A mutation known to block in vitro oligomerization of Rev abolished both complex formation and inhibitory activity of the mutant fusion proteins. Thus, trans-dominant inhibition of Rev does not require competition for nuclear substrates but may instead reflect the ability of a mutant to form nonfunctional complexes with the wild-type protein in vivo.

Binding, Competitive↗

PubMed1991

Effector domains of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex are functionally interchangeable and share an essential peptide motif.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex transactivators are posttranscriptional regulatory proteins that promote retroviral gene expression by interacting with specific viral mRNAs. Rev and Rex have markedly dissimilar amino acid sequences and RNA target specificities but are thought to act through the same cellular pathway. In this report, we demonstrate that short peptide domains which are required for effector activity in Rev and Rex are functionally interchangeable. Activity of these effector domains depends upon a previously unrecognized tetrapeptide motif that is present in both Rev and Rex and also in analogous proteins from other complex retroviruses. The conserved effector motif may mediate essential interactions of Rev, Rex, and other transactivators of this type with a common cellular cofactor.

Amino Acid Sequence↗