Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus

Abstract

We examined the kinetics and the nature of the association of two herpes simplex virus proteins, the major DNA-binding protein (ICP8) and the major capsid protein (ICP5), with the nuclei of infected cells. We defined a series of stages in the association of the ICP8 protein with the cell nucleus. (i) Immediately after synthesis, the protein was found in the cytoplasmic fraction but associated rapidly with the crude nuclear fraction. (ii) The initial association of ICP8 with the crude nuclear fraction was detergent sensitive but DNase resistant, and, thus, the protein was either bound to structures attached to the outside of the nucleus and had not penetrated the nuclear envelope or was loosely bound in the nucleus. (iii) At intermediate times, a low level of an intermediate form was observed in which the association of ICP8 with the nuclear fraction was resistant to both detergent and DNase treatment. The protein may be bound to the nuclear matrix at this stage. Inhibition of viral DNA synthesis caused the DNA-binding protein to accumulate in this form. (iv) At late times during the chase period, the association of ICP8 with the cell nucleus was resistant to detergent treatment but sensitive to DNase treatment. Our results argue that at this stage ICP8 was bound to viral DNA. Thus, nuclear association of the DNA-binding protein did not require viral DNA replication. More important is the observation that there is a series of stages in the nuclear association of this protein, and, thus, there may be a succession of binding sites for this protein in the cell during its movement to its final site of action in the nucleus. The major capsid protein showed some similar stages of association with the cell nucleus but the initial association with the nucleus followed a lag period. Its early association with the crude nuclear fraction was also detergent sensitive but was resistant to detergent treatment at later times. Its association with the cell nucleus was almost completely resistant to DNase treatment at all times. Inhibition of viral DNA replication blocked the nuclear transport of this protein. Thus, these two viral proteins share some stages in nuclear transport, although their requirements for nuclear association are different.

A major heat-shock protein defined by a monoclonal antibody.

“…This was vortexed gently, centrifuged at 2000 r.p.m. and repeated two more times when the preparation was 95% free of contaminating cytoplasmic proteins (Knipe and Spang, 1982). Purified nuclei were used to extract high salt-insoluble, nuclease-resistant nuclear structures essentially as described by Levinger and Varshavsky (1981).…”

Detection of herpes simplex virus proteins in cultured cells by monoclonal antibodies and the avidin‐biotin‐immunoperoxidase complex method

“…The predominant staining of ICP8 was the nuclear staining at 24 h alter infection, by which time the cytoplastnic ICP8 was not seen. Both the a and b forms of ICP8 ate present iti the cytoplastn and nucleus of infected cells (17,19). Although thete is no posttranslocational modification of 1CP8, the fact that this tnonoclonal antibody recognized the antigenie detertninant site of ICP8 protein suggests that both forms of ICP8 have the satne precursor and that this antigenie detettninant is maintained befote and alter ttanslocation.…”

“…The translocation of ICP8 ftotn the cytoplasm into the nucleus was seen at 8 h post-infection. Previous teports (18)(19)(20) demonsttated that imtnediately after ICP8 is synthesized, it is associated with the cytoplastnic framework; subsequently, it is transferred onto the nuclear Itatnework. However, its function in DNA replication is not clear.…”

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8

“…HSV-1 strain KOS 1.1 (13) was obtained from Myron Levine, University of Michigan, Ann Arbor. The virus was grown and titrated as described previously (15).…”

“…The fractions were dialyzed against 150 mM NaCl, precipitated with acetone, and dissolved in protein gel sample buffer. Equal portions of each fraction were analyzed by electrophoresis in polyacrylamide gels, followed by autoradiography (15).…”