Some characteristics of an early protein (ICP 22) synthesized in cells infected with herpes simplex virus

In Vero cells incubated at 40 degrees C or treated with azetidine at 37 degrees C, synthesis of a polypeptide ('C') of apparent mol. wt. 66000 was stimulated. It was not phosphorylated and was found in the cytoplasmic fraction of cell lysates. In cells infected with herpes simplex virus type 1 (HSV-1) in the presence of azetidine, synthesis of cellular proteins, including polypeptide C, was suppressed and infected cell polypeptides ICP 4, 0, 22 and 27 (apparent mol. wt. 170000, 120000, 75000 and 60000, respectively) were made. All were phosphorylated and accumulated in the nucleus. Messenger RNA for the same four polypeptides was made in cells infected in the presence of cycloheximide. Thus, ICP 22 is distinct from cellular polypeptide C and is probably a virus-specific alpha polypeptide, although it differs from alpha ICP 4, 0 and 27 in that its rate of synthesis does not decline rapidly when later polypeptides are produced. It is modified after synthesis in at least two steps, the second of which may require a later virus-specific polypeptide. In cells infected with HSV-2 the synthesis of a polypeptide analogous to ICP 22 could not be detected.     

The synthesis of the major DNA-binding protein (ICP8) in cells infected with the strain HSZP or KOS of herpes simplex virus type 1.

Synthesis of the major DNA-binding protein (ICP8) was investigated in primary rabbit kidney (RK) and Vero cells infected with the syncytial (syn) strain HSZP or with the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: 1. In contrast to strain KOS, the rate of viral polypeptide synthesis was accelerated in Vero cells infected with strain HSZP. The ICP8 could be detected in the nuclei of cells by one hour post-infection (hr p. i.) where it became associated with the viral DNA (DNase sensitive form). Later on (7 hr p.i.), the synthesis of viral polypeptides decreased and no further translocation of ICP8 from the cytoplasm into the nucleus was observed. 2. Strain HSZP was approx. three times more resistant to the action of phosphonoacetic acid (PAA) than strain KOS. In order to block the synthesis of HSZP gamma-2 polypeptides, a concentration of 600 micrograms PAA/ml had to be used. Under this condition, the HSZP ICP8 was translocated into the cell nucleus at later interval only (7 hr p.i.), and it was still possible to release this polypeptide from the nucleus by DNase treatment. The failure of the HSZP ICP8 to associate with the nuclear matrix (DNase resistant form) of infected cells in the absence of viral DNA replication may reflect its predominant affinity for the viral DNA which, in turn, may be responsible for the observed accelerated synthesis of the HSZP polypeptides in infected Vero cells. 3. In primary RK cells infected with strain HSZP the ICP8 did not translocate into the cell nucleus. Therefore, no gamma-2 polypeptides were synthesized.     

Comparative examination of the polypeptides of herpes simplex virus: types 1 and 2

Polypeptides synthesized during productive infection of HSV-1 and HSV-2 were found to possess distinct characteristics in regard to localization within the cell, DNA-binding properties, and phosphorylation after synthesis. Continuous labeling for 14 hr or pulse-labeling at successive periods during the replicative cycle with radioactive precursors revealed two types of polypeptide localization: (a) selective accumulation or enhancement within the cytoplasm or nucleus with barely detectable concentrations elsewhere and (b) accumulation in significant concentrations within both cytoplasm and nucleus showing little selective enhancement. Of the polypeptides made during HSV-1 infection 22 were phosphorylated as compared with 16 phosphoproteins specified by HSV-2. Phosphorylation was also implicated in the generation of the four molecular forms comprising the ICP 5–8 complex. Twenty-three polypeptides with affinity for DNA were detected after either type of infection. Sufficient comparisons were made to provide a basis for the tentative listing of 20 polypeptides of HSV-1 with corresponding polypeptides of HSV-2.     

Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells

Summary We have shown that a latent infection of herpes simplex virus type 2 (HSV-2) can be established in a human neuroblastoma cell line IMR-32 if the infected cells are cultured at 40°C. In the present study, viral polypeptides and cellular heat-shock proteins which were synthesized in HSV-2 infected IMR-32 cells cultured at 40°C were analyzed by polyacrylamide gel electrophoresis. It was found that the synthesis of late viral polypeptide ICP 5 was markedly reduced in the infected cells at 40°C as compared with those at 37°C. Although infection of IMR-32 cells with HSV-2 at 40°C resulted in shutoff of cellular protein synthesis, it was found that some cellular heat-shock proteins (90, 72 and 70 kd polypeptides) were synthesized and accumulated intracellularly. These findings suggest that modification of cascade regulation of HSV-2 polypeptide synthesis and/or accumulation of heat-shock proteins may be involved in the incomplete arrest of virus growth and in survival of the infected cells, leading to the establishment of HSV-2 latency in IMR-32 cells.     

On the association of virus proteins with the nuclei of cells infected with herpes simplex virus.

In cells infected with herpes simplex virus, HSV-I, newly synthesized polypeptides accumulated in the nucleus at different rates, which did not change during the first 6 h after infection. Canavanine, an arginine analogue, prevented the nuclear accumulation of ICP (infected cell polypeptides) 5 and 8 and azetidine, a proline analogue, prevented that of ICP 5 and 7. The transfer of polypeptides to the nucleus was inhibited at 4 degrees C but not by dinitrophenol. Some of the nuclear polypeptides could be released by washing isolated nuclei with hypertonic salt solutions. ICP 17 was particularly sensitive to high salt treatment while ICP 5 and II were resistent. ICP 4b, a modified form of the alpha polypeptide ICP 4, was released by EDTA, and the detergent NP40 removed ICP II. Treatment of nuclei with DNase selectively reduced the amount of bound alpha polypeptides ICP 4c (the second modified form of ICP 4), 0 and 27 as well as ICP 8 and 25. Nuclei isolated from infected or uninfected cells and incubated in labelled cytoplasmic extracts took up primarily ICP 8 and 32. Alpha polypeptides were taken up to a lesser extent and ICP 6 and 10 were excluded. It is concluded that affinities for various constituents of host cell nuclei are likely to determine the nuclear accumulation of specific virus polypeptides.     

Host cell and virus strain differences in synthesis of immediate early polypeptides in HSV-1 infected cells

The synthesis of the immediate early (IE) polypeptides was analysed in primary rabbit kidney (RK) cells and a stable line of rabbit lung (ZP) cells infected with the syncytial (syn) strain HSZP and the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: After cycloheximide reversal the infection of RK and ZP cells with HSZP strain led to synthesis of five IE polypeptides (175K, 136K, 87K, 68K, and 63K), while infection of both cell cultures with the KOS strain led to synthesis of significantly reduced amounts of the IE polypeptides. The ability to switch on the expression of non-alpha viral genes was impaired in RK cells infected with the HSZP strain. The IE polypeptides were still detectable without any sign of the non-IE polypeptide synthesis 4 hr after cycloheximide reversal. The observed failure of the IE HSZP polypeptides to undergo posttranslational modification in ZP cells may be the consequence of this phenomenon. In contrast to the KOS IE mRNAs, the HSZP IE mRNAs exhibited a pronounced functional stability in both cell cultures. The IE polypeptides were still synthesized in HSZP-infected cells which had been incubated for 19 hr after cycloheximide reversal in the presence of actinomycin D (Act D). The HSZP strain failed to suppress the host polypeptide synthesis in RK but not in ZP cells. However, the HSZP strain, in contrast to the KOS strain, proved to be defective with respect to the early shutoff of host polypeptide synthesis in both cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells

Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons.     

The Herpes Simplex Virus Type 1 Transactivator ICP0 Mediates Aberrant Intracellular Localization of the Viral Helicase/Primase Complex Subunits

The infected cell polypeptide 0 (ICP0) protein of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator. When expressed by transfection, ICP0 forms spherical structures in the nucleus. Using a double-label immunofluorescence assay, we have found that the HSV-1 helicase/primase complex subunits accumulate within ICP0 structures in cotransfected cells. This phenomenon was also observed in cells coexpressing ICP0 and UL6, a protein thought to be involved in the cleavage and/or packaging of viral genomes. ICP0 structures were found to be proteinaceous by immunoelectron microscopy. These results suggest that ICP0 may interact nonspecifically with a variety of viral proteins.     

Immunological reactivity of herpes simplex virus 1 and 2 polypeptides electrophoretically separated and transferred to diazobenzyloxymethyl paper.

In this paper we report that viral polypeptides from herpes simplex virus 1 (HSV-1) and 2 (HSV-2)-infected cells electrophoretically separated in sodium dodecyl sulfate-polyacrylamide-agarose gels and transferred to diazobenzyloxymethyl paper can react with rabbit hyperimmune sera, both polyvalent and prepared against specific antigens. The polyvalent hyperimmune sera against HSV-1 reacted with 17 HSV-1 polypeptide bands and 8 HSV-2 polypeptide bands. Concordantly, polyvalent sera against HSV-2 reacted with at least 16 HSV-2 polypeptide bands and 8 HSV-1 polypeptide bands. The antisera prepared against the specific antigens reacted with a smaller number of polypeptide bands. Preimmune sera and immune sera did not react with electrophoretically separated polypeptides from infected and uninfected cells, respectively. The immune localization of separated antigens test provides a powerful technique for identification of immunogenic viral polypeptides, especially those which are normally insoluble and therefore unavailable for immunological reactivity in immune precipitation tests.     

Structure of the Mengo virion. V. Distribution of the capsid polypeptides with respect to the surface of the virus particle.

The disposition of the Mengo capsid polypeptides (α, β, γ, and δ) with respect to the external surface of the virion has been investigated by measuring their relative susceptibilities to lactoperoxidase-catalyzed iodination and their reactivities in immunological tests with specific antisera. When intact virions were subjected to iodination for a brief period of time (1 min), radioactive iodine was incorporated predominantly into the a polypeptides and to a lesser extent into β polypeptides. Only with longer incubation times (15 min or more) did label appear in the γ and δ polypeptides; and this coincided with a progressive loosening and ultimate collapse of the viral capsid. Antisera specific for each of the capsid polypeptide species were produced in rabbits using isolated proteins as antigens. Reaction of virions with these antisera in plaque-neutralization and hemagglutination-inhibition tests showed that only the anti-α serum was capable of blocking virus-cell interactions. Complement-fixation and immunodiffusion tests confirmed the observations that the α polypeptides occupy most of the external surface of the virus particle and that the β polypeptides are partially exposed. The γ and δ polypeptides apparently occupy internal locations in the capsid of the Mengo virion.     

Proteins of herpesvirus type 2. II. Studies demonstrating a correlation between a tumor-associated antigen (AG-4) and a virion protein.

Antigen AG-4, a HSV-2 antigen associated with actively growing squamous cervical tumors, correlates with infected cell protein number 10 (ICP 10), a minor component of the HSV-2 virion. This association is based on the following evidence: (i) There is a positive correlation between the amounts of AG-4 and ICP 10 produced at various times following sequential treatment of infected cells with cycloheximide and actinomycin-D; (ii) the passage history of HSV-2 affects the synthesis of AG-4 and ICP 10 in a similar manner; their synthesis is unaffected by the cell type; (iii) ICP 10 is precipitated by AG-4 positive but not by AG-4 negative sera; and (iv) following partial biochemical purification of crude AG-4 preparations, those fractions containing the AG-4 complement-fixing activity differ from those without this activity in that they contain ICP 10.     

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC₄) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC₄ cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC₄ cells was enhanced 2.6±0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC₄ cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC₄ cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.     

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.     

Transactivation by herpes simplex virus proteins ICP4 and ICP0 in vaccinia virus infected cells.

Vaccinia virus recombinants containing the sequences from herpes simplex virus type 1 (HSV-1) encoding the immediate early (IE)(alpha) proteins ICP4 and ICP0, under the control of a mutated vaccinia virus 11K late promoter, were constructed. A cDNA copy of the gene encoding ICPO and an ICP4-encoding genomic segment were each inserted into the vaccinia virus genome at the thymidine kinase (TK) locus by homologous recombination. Steady-state analyses revealed that RNAs homologous to the IE-0 and IE-4 sequences accumulated in cells infected by recombinants with the kinetics of a typical vaccinia late mRNA. Western blot analyses demonstrated that the expression level of both ICPO and ICP4, produced by the recombinant viruses, was comparable to that in HSV-1-infected cells at late times postinfection. Both proteins synthesized in cells infected by the recombinants were located in the nucleus as revealed by immunofluorescence. Although in vitro studies reveal that extracts from vaccinia-virus-infected cells lose the ability to transcribe genes that contain RNA polymerase II promoters (Puckett and Moss (1983), Cell 35, 441-448) both ICPO and ICP4 expressed by the recombinant viruses can transactivate plasmids containing a reporter gene driven by the promoters for the HSV-1 TK and glycoprotein C genes. Nuclear extracts prepared from cells infected with the vaccinia virus vector expressing ICP4 exhibited sequence-specific DNA-binding activity.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

Analysis of the HSV-2 early AG-4 antigen

Summary Genital herpes simplex virus type 2 (HSV-2) infections can be distinguished from present or past HSV-1 infections by an AG-4 antigen complement fixation assay. The assay which utilizes a 4 hour HSV-2 infected cell extract prepared at a multiplicity of infection (MOI) of 1.0 PFU/cell, appears to consist of several viral proteins. Studies using monoclonal antibodies, polyclonal rabbit hyperimmune serum, HSV-1×HSV-2 intertypic recombinant viruses and polyacrylamide gel electrophoresis suggest that ICP8 may be one of the major antigens involved in the complement fixing reaction. It is probable that the success of the assay is not due to a true type specificity but rather a threshold phenomenon in which HSV-2 extracts contain more early viral antigens (including ICP8) and sera from HSV-2 patients contain more complement fixing antibody to these antigens.With 4 Figures     

Characterization of the large tegument protein (ICP1/2) of herpes simplex virus type 1.

ICP1/2 (also designated VP1/2) is a 270-kDa structural protein of herpes simplex virus type 1 (HSV-1) which is located in the tegument region of the virion. In this report we describe the production of a polyclonal antiserum specific for ICP1/2 and the use of this antiserum to examine the synthesis, processing, and intracellular localization of the viral polypeptide. Pulse-labeling studies indicated that ICP1/2 is synthesized late during infection, being initially detectable between 8 and 9 hr postinfection with the rate of synthesis continuing to increase until 11 to 12 hr postinfection. Further studies on the expression of ICP1/2 in the presence or absence of viral DNA replication indicated that the synthesis of the polypeptide is absolutely dependent on viral DNA replication. These results suggest that ICP1/2 represents a gamma 2 (true late) gene product. Additionally, we have performed experiments to determine if ICP1/2 is post-translationally modified in HSV-infected cells. These studies indicated that ICP1/2 is phosphorylated on serine residues; however, we found no evidence to suggest that the protein is glycosylated. Using subcellular fractionation and indirect immunofluorescence techniques, we have determined that ICP1/2 is diffusely distributed throughout the nucleus and cytoplasm of HSV-infected cells with no specific compartmentalization of the polypeptide.     

The cell-free synthesis of herpesvirus-induced polypeptides.

Translation of cytoplasmic RNA from herpes simplex virus (HSV)-infected BHK cells has been achieved by the use of a cell-free system derived from mouse and rabbit reticulocytes. Protein synthesis was not significantly enhanced by addition of a ribosomal salt-wash fraction prepared from HSV-1-infected cells, whereas an equivalent fraction from uninfected cells stimulated equally the synthesis of HSV-induced, BHK cell, and reticulocyte polypeptides. The polypeptides produced in vitro in the presence of cytoplasmic RNA from HSV-1, HSV-2, and a deoxypyrimidine kinaseless mutant of HSV-1 show many of the electrophoretic mobility differences observed for infected cell polypeptides, suggesting that the accurate synthesis of many unmodified viral polypeptides occurs in the cell-free system.     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.

Several putative NF-kappa B-binding sites in the ICP0 and Vmw65 herpes simplex virus type-1 (HSV-1) genes have been identified. Oligonucleotides encoding some of these sites bind specifically to purified NF-kappa B protein and an NF-kappa B-like protein in nuclear extracts of phorbol ester- or cycloheximide-induced human embryonic lung (HEL) cells. HSV-1 infection of HEL cells induced a nuclear factor that binds specifically to kappa B sites in the ICP0 and Vmw65 gene regions and comigrates with complexes formed by purified NF-kappa B. The HSV-1-inducible nuclear factor bound to the authentic immunoglobulin (Ig) kappa B site. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing two copies of the Ig kappa B site upstream of the c-fos promoter (kappa B2-CAT) showed activity in HEL cells. HSV-1 infection of kappa B2-CAT-transfected HEL cells, however, induced a dramatic increase in CAT activity; mutation in the NF-kappa B-binding site of kappa B2-CAT abolished the inducibility of CAT gene expression. Our results demonstrate that the HSV-1 ICP0 and Vmw65 gene regions contain binding sites for NF-kappa B, and that HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.