Laure Aurelian

THE MULTIPLICATION OF HERPES SIMPLEX VIRUS. I. THE PROGRAMMING OF VIRAL DNA DUPLICATION IN HEP-2 CELLS.

Abstract This paper reports the results of two series of experiments. In one series it was estimated with the aid of 5-iodouracildeoxyriboside (IUdR) that in HEp-2 cells, Herpes simplex virus (HSV) DNA accumulates at an exponential rate between 4 and at least 14 hours after infection. The time required to duplicate one molecule of viral DNA cannot be more than 40 minutes; the time interval between completion of DNA and its encapsidation is estimated to be 120–150 minutes. In the second series, infected cells were exposed to IUdR or to puromycin for brief intervals of time. IUdR added to cells for brief intervals had no demonstrable effects during the first 2 hours. In cells exposed to IUdR during the third and fourth hours virus multiplication was delayed, but the rate of accumulation of virus was unaffected. However, the inhibition of virus multiplication in cells treated for 2 hours between 4 and 14 hours after infection could not be reversed. Incorporation of IUdR-H3 into viral DNA could not be shown. Brief treatment of infected cells with puromycin during the first 4 hours of infection delayed, but did not affect the rate of, viral DNA synthesis and virus maturation. The inhibition produced later in the infection could be reversed by withdrawing the drug. These results are interpreted as follows: (1) Protein synthesis must precede viral DNA synthesis. (2) The enzymes required for the utilization of thymidine in the synthesis of viral DNA are made approximately between the second and fourth hours after infection. The synthesis of these enzymes ceases after the onset of viral DNA synthesis. (3) The duplication of viral DNA begins when one or more specific precursors of DNA synthesis reach a critical concentration.

Herpesvirus Type 2 Isolated from Cervical Tumor Cells Grown in Tissue Culture

A herpesvirus has been isolated from spontaneously degenerating cultures of cervical tumor cells grown in vitro. The virus was identified as a type 2 herpesvirus on the basis of biologic properties, including plaque morphology and microtubule formation in infected HEp-2 cells, and of immunologic specificity as determined by neutralization. Herpesvirus antigens and virus particles were not seen in duplicate cultures of viable cervical tumor cells.

Antibody to HSV-2 Induced Tumor Specific Antigens in Serums from Patients with Cervical Carcinoma

Antibody distinct from that involved in neutralization and directed to an antigen (AG-4) induced in HEp-2 cells by infection with herpesvirus type 2 was identified in serums from patients with cervical carcinoma by means of a quantitative micro complement fixation test. The presence of antibody to AG-4 correlates well with the extent of the tumor; antibody is virtually absent in matched control women and in women with therapy and without recurrent neoplasia. Reactivity is not observed with control antigen consisting of a cell extract prepared from uninfected HEp-2 cells. The possible prognostic significance of this antibody and its implications are discussed.

Abortive infection of canine cells by herpes simplex virus: I. Characterization of viral progeny from co-operative infection with mutants differing in capacity to multiply in canine cells*

The conditional lethal mutant MP dk − of herpes simplex virus multiplies in cells of human derivation. In dog-kidney cells the virus produces ENA and antigen but no infectious progeny (Aurelian & Eoizman, 1964) or particles with physical properties of herpes-virus virions. Continuous propagation of infected dog-kidney cells for intervals as long as 10 weeks yielded small (MP dk + sp ) and large (MP dk + lp ) plaque variants capable of multiplying in both human and canine cells. The MP dk + sp and MP dk + lp mutants differ from MP dk − with respect to plaque morphology in human cells, buoyant density in cesium chloride solution, stability at 40°C and reactivity with rabbit anti-MP dk - serum. Simultaneous infection of dog-kidney cells with both MP dk + sp and MP dk − yielded infectious progeny consisting of virions with the genotype of either strain but with the phenotype of MP dk + sp only. It is concluded that, in dog-kidney cells, one or more structural constituents of the MP dk − virion are either not made or they are non-functional.

The Host Range of Herpes Simplex Virus. Interferon, Viral DNA, and Antigen Synthesis in Abortive Infection of Dog Kidney Cells.

Abstract Herpes simplex virus strain MP (HSV-MP) and pseudorabies virus (PSV) multiply in HEp-2 cells. In dog kidney cells PSV multiplies, whereas HSV-MP fails to multiply or to produce plaques. HSV-MP interferes with the multiplication of PSV in HEp-2 cells and in dog kidney cells. Studies of dog kidney cells exposed to HSV-MP revealed that these cells (1) produce viral antigen, interferon, and small amounts of DNA characteristic of HSV-DNA, (2) do not form particles with physical characteristics of HSV-MP, and (3) show signs of infection and fail to multiply. The significance of the data is discussed.

ABORTIVE INFECTION OF CANINE CELLS BY HERPES SIMPLEX VIRUS. II. ALTERNATIVE SUPPRESSION OF SYNTHESIS OF INTERFERON AND VIRAL CONSTITUENTS.

Abortive infection of dog-kidney cells with herpes simplex virus strain MP dk − yields viral DNA and antigen, and interferon, a host response to infection (Aurelian & Roizman, 1964). In the present paper it is shown that the production of viral constituents and interferon are alternatively suppressed; viral constituents are synthesized in cells infected at multiplicities of 100 or more plaque-forming units/cell, whereas peak interferon production takes place in dog-kidney cells infected with 10 plaque-forming units/cell. Productive infection of dog-kidney cells with host-range mutant MP dk + sp (Roizman & Aurelian, 1965) yields infectious progeny but no interferon. The basis for the suppression of interferon synthesis in MP dk − -infected dog-kidney cells producing viral constituents became apparent from studies of cellular RNA synthesis following abortive infection with MP dk − and productive infection with MP dk + sp . Thus, at multiplicities of 12 MP dk + sp plaque-forming units per dog-kidney cell, RNA synthesis rapidly decreases. In cells infected at similar multiplicities with MP dk − , the decrease in RNA synthesis is delayed until 5·5 hours after infection. At high multiplicities of infection, the pattern of RNA synthesis approaches that seen in cells infected with MP dk + sp . It is concluded, therefore, that at high multiplicities of infection, rapid decrease in host macromolecular metabolism precludes interferon synthesis; at low multiplicities, the host responds to the presence of foreign matter before the macro-molecular metabolism is altered by the virus.

Antiviral effect of oligo(nucleoside methylphosphonates) complementary to the herpes simplex virus type 1 immediate early mRNAs 4 and 5

We have previously shown that an oligo(nucleoside methylphosphonate) (deoxynucleoside methylphosphonate residues in italics) complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) immediate-early (IE) pre-mRNAs 4,5 [d(TpTCCTCCTGCGG)], causes sequence-specific inhibition of virus growth in infected cell cultures (Smith et al., 1986; Kulka et al., 1989). Here we report a similar inhibition of HSV-1 growth by oligo(nucleoside methylphosphonates) complementary to the splice donor site of HSV-1 IE pre-mRNAs 4,5 [d(GpCTTACCCGTGC)] and to the translation initiation site of IE4 mRNA [d(ApATGTCGGCCAT)]. An oligomer complementary to the translation initiation site of IE5 mRNA [d(GpGCCCACGACAT)] or an unrelated oligomer [d(GpCGGGAAGGCAC)] did not inhibit virus growth. IC50 values were 20, 25 and 20 microM for d(TpTCCTCCTGCGG), d(GpCTTACCCGTGC) and d(ApATGTCGGCCAT) respectively. In infected BALB/c mice d(TpTCCTCCTGCGG) caused a significant decrease in HSV-1 growth (82% inhibition at 500 microM). A psoralen-derivative of d(TpTCCTCCTGCGG) that binds covalently to complementary sequences after exposure to 365 nm irradiation, inhibited HSV-1 growth (86-91%) at a 10-fold lower concentration than the non-derivatized oligomer. The inhibition was sequence-specific and significantly lower (27%) for HSV-2 that differs from HSV-1 in 7 of the 12 bases targeted by d(TpTCCTCCTGCGG). Virus growth was not inhibited by d(GpGCCCACGACAT). The data suggest that oligo(nucleoside methylphosphonates) may be effective antiviral agents.

Myristylation and polylysine-mediated activation of the protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10)

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) was previously shown to possess protein kinase (PK) activity that localizes to the cytosolic, cytoskeletal, and plasma membrane fractions. Further studies of the PK domain using computer-assisted sequence analysis have identified a single transmembrane segment and fatty acid incorporation findings indicate that ICP10 is myristylated. Myristylation does not depend on a viral enzyme, since myristic acid is incorporated into ICP10 precipitated from cells transfected with an ICP10 expression vector. It is also incorporated into the 57-kDa protein expressed by the amino-terminal PK expression vector. The myristyl moiety is linked through an amide bond. The basic protein polylysine stimulates the kinase activity and alters its divalent cation requirements resulting in 20- to 40-fold stimulation in the presence of 0.1 mM Mn2+. The PK activity is inhibited by antibody to synthetic peptides corresponding to residues 355-369 and 13-26, respectively, located within, and amino-terminal to, the predicted PK catalytic domain.

Viruses and gynecologic cancers: Herpesvirus protein (ICP 10/AG‐4), a cervical tumor antigen that fulfills the criteria for a marker of carcinogenicity

The studies associating infections by herpes simplex virus type 2 (HSV‐2) with carcinoma of the human uterine cervix are reviewed within the context of three possible interpretations. Extensive seroepidemiologic evidence indicates that the virus does not grow preferentially in neoplastic tissue, nor is the association of HSV‐2 with cervical carcinoma a reflection of their independent relationship to promiscuity. While a number of infectious agents, including other viruses, are associated with cervical atypia, only HSV‐2 is a significant risk factor for CIS. In vitro transformation data supporting the oncogenic potential of the virus are summarized, and evidence is presented that an antigen designated ICP 10/AG‐4 is a valid candidate for the role of a virus‐encoded protein involved in the maintenance of a transformed phenotype. Antibody to AG‐4 is IgM, and it is detected by microquantitative complement fixation. With this assay, it is demonstrated that conversion to anti‐AG‐4 occurs during primary infection with HSV‐2; however, it is transient. In testing 1325 patients, a correlation was observed between antibody to AG‐4 and cervical carcinoma. Thus, whereas only 11.7% of controls and 7.7% of patients with cancer at other sites are AG‐4 seropositive, as many as 49.6% of patients with dysplasia, 63% of those with CIS, and 72.7% of those with invasive cancer are positive for the antibody. Antibody to AG‐4 is related to tumor growth. This is evidenced by 1) retrospective analyses demonstrating that the proportion of AG‐4 seropositive individuals is directly correlated to the stage of the disease and 2) prospective study of 209 patients demonstrating loss of antibody in patients with a successfully removed tumor mass and reappearance of AG‐4 antibody in cancer recurrence. The possible use of AG‐4 (or its antibody) in the diagnosis and monitoring of cervical carcinoma and its treatment is discussed.

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

Abstract 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.