The immune response to herpes simplex virus: comparison of the specificity and relative titers of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections

Employing an immunoblotting technique, the polypeptide specificity and relative titers of anti-HSV IgG reactive with denaturation-resistant epitopes on HSV proteins were determined in patients experiencing primary HSV-1 infections at various anatomical sites. Early sera from previously seronegative patients with primary HSV-1 infections were found to have comparatively low levels of antibody directed against the major viral glycoprotein antigens (gB, gC, and gD) relative to titers present in sera of individuals with long-standing, latent orofacial HSV-1 infections. Patients with primary infections did however have high titers of antibody directed against a series of low molecular weight HSV polypeptide antigens. These antigens were found to be antigenically related to a structural component of virion nucleocapsids. At later times postinfection, titers of antibodies directed against other viral polypeptides including the major glycoproteins increased to levels more closely approximating those observed in latently infected individuals. These results indicate that the anti-HSV IgG detected by immunoblot analysis which appears earliest following primary infection is not directed against the known major infected cell or virion glycoprotein surface antigens but rather against an internal capsid protein of HSV.     

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.     

Detection of an immediate early herpes simplex virus type 1 polypeptide in trigeminal ganglia from latently infected animals.

In this study, trigeminal sensory ganglia from animals with acute herpes simplex virus, type 1 (HSV-1) infection were compared to those with a latent infection for the expression of HSV-specific antigens. By the indirect immunofluorescence assay, antisera to an immediate early polypeptide of molecular weight 175,000, designated VP175 or ICP4, and a hyperimmune antiserum to HSV-1 were used to determine whether early viral polypeptides were being expressed in neurons during the latent stage of infection. All 17 ganglia from animals with acute infection (sacrificed 3 to 12 days postinfection) exhibited positive staining when treated either with anti-HSV-1 or with anti-VP175. Forty of 42 ganglia from animals sacrificed during the latent stage of infection (22 to 200 days postinfection) exhibited immunofluorescent staining when treated with anti-VP175. The staining appeared to be similar to that observed in ganglia from acutely infected animals stained with anti-VP175, except that the number and distribution of stained cells were markedly reduced. No immunofluorescence was observed in ganglia from noninfected control animals when stained with anti-VP175 or anti-HSV-1, or when ganglia from latently infected animals were stained with anti-HSV-1 or preimmune serum.     

The isolation of Mengo virus stable non-capsid polypeptides from infected L cells and preliminary characterization of an RNA polymerase activity associated with polypeptide E.

Isolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS. Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.     

Antibody responses in humans to individual proteins of herpes simplex viruses.

Sera from 231 women were used to examine their frequency of precipitation of various herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) proteins and to determine if there was a rank order of immune responsiveness of humans to these HSV antigens. Radiolabeled viral proteins were reacted with serum and immune complexes isolated with staphylococcal protein A. Individual antigens were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. As a group, these sera precipitated 31 HSV-1 and 27 HSV-2 proteins. HSV-1 polypeptides with molecular weights of 133,000, 99,000, and 82,000, as well as HSV-2 polypeptides with molecular weights of 131,000 and 101,000, were precipitated by essentially all sera that contained antibodies to HSV-1 and HSV-2. When attempts were made to order the viral proteins by constructing precipitation profiles ranking the antigens in patterns according to their frequency of precipitation, it was observed that the antigens were generally not ordered. Demographic analysis of the sera suggested that the differences in the number of proteins precipitated were associated with differences in age, education, age at first marriage, and income, which collectively may reflect the frequency of exposure to the virus.     

Human antibody response to herpes simplex virus-specific polypeptides after primary and recurrent infection.

Human antibody responses to specific polypeptides of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) were assessed in serial serum specimens from 18 infected patients by immunoblot technology. Nine patients had HSV-1 infections (six genital and three oral) and nine had HSV-2 genital infections. Antibodies to homologous and heterologous HSV antigens were studied and correlated with total microneutralization and enzyme-linked immunosorbent assay antibodies as well as correlated directly to purified glycoproteins. The data indicated a sequential appearance of antibodies to specific polypeptides, according to virus type and site of infection. After HSV-1 infection, the initial response was to glycoprotein B, but the same was not true for HSV-2 infection, where the initial response appeared to be to the type-specific glycoprotein G. A difference in sequential appearance of antibodies for the two viruses indicated greater reactivity to lower-molecular-weight polypeptides after genital infection, irrespective of type, in contrast to nongenital HSV-1 infections. The antibody responses for selected sera to purified glycoproteins B and D were verified by enzyme-linked immunosorbent assay antibody determinations.     

Immunoprecipitation of polypeptides from hamster embryo cells transformed by herpes simplex virus type 2

Hyperimmune rabbit antisera to herpes simplex virus type 2 (HSV-2) were used to immunoprecipitate proteins from extracts of hamster cells tranformed by HSV-2. Eight polypeptides of molecular weights 110K, 84K, 77K, 47K, 38K, 37.5K 35K, and 32K were precipitated from transformed (620-7, 620-14) or tumor-derived cells (620-7T) by anti-HSV serum but not by control serum. These polypeptides were not precipitated from adenovirus-transformed hamster or hamster embryo fibroblast cells. Three additional polypeptides of molecular weights 80K, 66K, and 29.5K could be detected when high salt (1.4 M NaCl) was not employed in the immunoprecipitation procedure.     

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     

Reovirus-coded polypeptides in infected cells: Isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively

The cytoplasm of cells infected with rovirus contains 8 of the 10 polypeptides coded by this virus in free form in amounts equivalent to 1 to 10% of the progeny virus yield. All of these polypeptides exist in monomeric form except polypeptides μ1 and μ2 which occur as dimers, the form in which they are present in virions. One of these polypeptides, σ3, a constituent of the outer capsid shell, binds strongly to double-stranded RNA; it can be isolated in high yield in pure form by passing extracts of infected cells through columns of poly(IC)-cellulose and eluting. Another polypeptide, σ2A, a nonstructural polypeptide, binds strongly to single-stranded RNA and appears to exist in infected cells complexed with RNA. It can be isolated in high yield in pure form by passing extracts of infected cells, suitably treated to dissociate these complexes, through columns of poly(A)-cellulose and eluting. The possible significance of the highly specific nucleic acid affinities of these two reovirus-coded polypeptides is discussed.     

Biochemical and serological properties of the thymidine-phosphorylating enzymes induced by herpes simplex virus mutants temperature-dependent for enzyme formation.

Evidence is presented in support of the hypothesis that: (i) an enzyme induced by wild-type herpes simplex virus type 1 (HSV-1) is a deoxypyrimidine kinase with a single active site for the phosphorylation of both thymidine (TdR) and deoxycytidine (CdR); (ii) enzymes induced by HSV-1 mutants that are temperature dependent for enzyme induction are altered with respect to nucleoside acceptor specificity and antigenicity; and (iii) altered polypeptides are synthesized in mutant virus-infected cells at both the permissive (31°) and restrictive (37.5°) temperatures. HSV-1 mutants B2010 and B2015 induce less TdR-phosphorylating activities at 31° than wild-type HSV-1 and very low but detectable enzyme activities at 37.5°. The TdR-phosphorylating activities induced by the mutant viruses at 31° have the same electrophoretic mobilities as that of the wild-type HSV-1-induced enzyme but, unlike the wild-type enzyme, the mutant enzymes lack CdR-phosphorylating activity. Immunoglobulin G (IgG) prepared from rabbits immunized with cytosol extracts from rabbit kidney cells infected with wild-type HSV-1 inhibit the TdR-phosphorylating activities induced by wild-type and mutant viruses, as well as the CdR-phosphorylating activity induced by wild-type HSV-1. Extracts purified from cells infected at 31° with wild-type and mutant HSV-1 blocked the neutralizing activity of anti-HSV-1 IgG for the CdR- as well as the TdR-phosphorylating activities of wild-type HSV-1 enzyme, despite the lack of CdR-phosphorylating activity of the mutant enzyme. However, the mutant virus-infected cell extracts were less effective than wild-type extracts in serum blocking activity. Extracts from cells infected with mutant B2015 at 37.5° did not exhibit serum blocking power.     

Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells.

Previous reports from this laboratory have defined as α those viral polypeptides whose production in infected cells does not require prior protein synthesis. Two subsequent groups, β and γ, depend on prior a and β polypeptide synthesis, respectively. Comparison of the synthesis and properties of a polypeptides specified by herpes simplex viruses (HSV) 1 and 2 showed the following: (i) The three earliest virus-specific infected cell polypeptides (ICP) made in HSV-2 infected cells migrated slightly more slowly in polyacrylamide gels than the HSV-1 α polypeptides, ICP 4.0 and 27. (ii) Cells treated with canavanine from the time of infection with HSV-2 produced all α, a subset of β, and a small amount of one γ polypeptide; the synthesis of these polypeptides continued for many hours, at rates related to the multiplicity of infection. The transition from a to this subset of β polypeptide synthesis did not appear to be affected by the arginine analogue, whereas transition to the other sets of β and to γ polypeptide synthesis was blocked. A similar discrimination between different subsets of β proteins was seen in treated HSV-1 infected cells. (iii) All a and a number of β polypeptides observed in this study underwent translocation into the nucleus, posttranslational modification resulting in a reduced electrophoretic mobility, and phosphorylation. For example, the modification of HSV-1 and HSV-2 ICP 4 was in at least two steps from the translational product ICP 4a, labeled during a pulse, to slower-migrating forms ICP 4b and 4c. All three forms were phosphorylated but only 4b and 4c were found in the nucleus. In untreated infected cells, ICP 4 ultimately accumulated in form ICP 4c. ICP 4a made in the presence of canavanine was not processed efficiently into ICP 4c. In another instance, the polypeptide made in the presence of canavanine were not translocated into nuclei.     

Assay of type-specific and type-common antibodies to herpes simplex virus types 1 and 2 in human sera.

A reliable and reproducible method for determining specific reactivity to herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in human sera has been developed. Human sera were used to immunoprecipitate HSV-specific glycoprotein antigens from both HSV-1- and HSV-2-infected cell extracts. The viral glycoproteins precipitated from these extracts were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect specific reactivity of the sera with distinct type-specific antigens of HSV-1, HSV-2, or both as well as with type-common glycoprotein antigens. By examining a large number of human sera, this method was found to be more reliable than the standard microneutralization test in discriminating between single-positive (positive for HSV-1 or HSV-2) and double-positive (positive for HSV-1 and HSV-2) sera.     

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.     

Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts

Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated paramagnetic Dynabeads (Dynal) has been used to isolate HSV-2 type-specific gG-2 from solubilized HEp-2 HSV-2 infected cell extracts. The immunomagnetically captured type-specific glycoprotein reacted strongly with monoclonal antibody H1206 and demonstrated a single band with apparent molecular weight of 100000 (100 kDa) and a doublet band with an apparent molecular weight of 60000-64000 (60-64 kDa). We observed the same exact banding pattern when monoclonal H1206 was immunoblotted with Helix pomatia lectin purified HSV-2 gG-2. The immunomagnetically purified gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1 and four human HSV antibody negative sera. In addition, 20 human HSV antibody positive sera obtained from the Centers for Disease Control (CDC), Atlanta, GA, were used for the evaluation of our methodology. Immunoblotting of the human HSV antibody positive samples were in agreement with the CDC HSV serological designation. Sera characterized by reactivity to the immunomagnetically purified gG-2 in conjunction with Western blot has the potential to be used as a confirmatory serological test or to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.     

Complement-mediated cytolysis of HSV-1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody.

Surface antigens of BHK-21 cells infected with HSV-1 or HSV-2 were radioiodinated (125I) with lactoperoxidase, immune precipitated and analysed by polyacrylamide gel electrophoresis (PAGE). Experiments using antiserum to HSV-1 or HSV-2, absorbed with appropriate hemotypic or heterotypic antigens, revealed that both type-specific (homotypic) and cross-reactive antibody combined with surface glycoproteins to form a single large radioactive peak. This peak, which constituted the major glycoprotein region (region a) observed in electropherograms, represented a range in mol. wt. from 115000 to 130000. Sensitization of cells to complement lysis, neutralization of infectious virus and immune precipitation of surface glycoproteins (region a) were found to be generally correlated properties of all the antibody preparations analysed, including antibody prepared specifically against region a antigens. These findings suggest a major immunological role for the surface glycoproteins migrating in PAGE region a.     

Type-specific identification of herpes simplex and varicella-zoster virus antigen in autopsy tissues.

To identify antigens of herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV) in human tissue, polyclonal antisera and an immunoperoxidase method were used to examine formalin-fixed, paraffin-embedded tissues from autopsy cases and experimentally infected animals. These antisera readily distinguished between HSV and VZV antigen, with no evident cross-reactivity. Antiser ato HSV-1 and HSV-2 were more strongly reactive with antigen of the homologous virus than with that of heterologous virus. This difference in immunoreactivity was used to discriminate between HSV-1 and HSV-2 antigens in experimentally infected animal tissues containing HSV antigens of known type and, by extrapolation, to distinguish between these antigens in human autopsy tissues. Thus, with appropriate antisera and tissue controls, HSV-1, HSV-2, and VZV can be identified in paraffin sections.     

ELISA for detection of IgG and IgM antibodies to HSV-1 and HSV-2 in human sera

A rapid, enzyme-linked immunoassay (ELISA) was applied to identify and measure specific IgG and IgM antibodies to herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2). Detergent solubilized infected cells and mock-infected cells were used as antigens in the assay. Identification of type-specific antibodies was achieved by a competition assay in which clinical sera mixed with HSV-1 or HSV-2 antigens were assayed for reactivity to identical antigens coating wells of polystyrene microtiter plates. Reactivity and the specificity of the reactive immunoglobulin class was quantitated using biotinylated goat anti-IgG and biotinylated goat anti-IgM. Five paired sera from patients with diagnosed herpes simplex genital infections and one human anti-HSV-1 reference serum were tested with this assay and results were compared to results previously obtained using a complement fixation test and micro-SPRIA. The results indicate that the ELISA is a specific, sensitive and simple test which confirms the herpes simplex virus infection history of patients.     

The 10K virion phosphoprotein encoded by gene US9 from herpes simplex virus type 1

Gene US9 of herpes simplex virus type 1 has been predicted, from DNA sequence analysis, to encode a protein of mol wt 10,026, designated 10K (D.J. McGeoch, A. Dolan, S. Donald, and F.J. Rixon (1985). J. Mol. Biol. 181, 1-13). We have investigated this protein by using a synthetic peptide corresponding to the 11 amino acids adjacent to the amino-terminal methionine and rasing antisera in rabbits. One antiserum was able to precipitate at least 12 electrophoretically distinct polypeptide species from extracts of BHK cells infected with HSV-1. The estimated molecular weights of these polypeptides ranged from 12K to 20K and immunoblotting showed them to be related proteins. The primary translation product has an apparent mol wt of 13K. The various forms of 10K differ in their relative abundance in the infected cell and also in their degree of phosphorylation. Lower molecular weight forms of the 10K protein can be precipitated from NP-40 extracts of HSV-1 virions, suggesting that these forms of 10K are contained in the virion tegument or envelope. An association between this protein and nucleocapsids has also been observed in the nuclei of infected cells by immunoelectron microscopy. These observations imply that the product of US9 is a tegument protein which becomes associated with nucleocapsids at, or soon after, their formation in the nuclei of infected cells.     

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.     

Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein

The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.