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Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice.

Viral and cellular factors responsible for parvovirus target cell specificity have been examined for two serologically indistinguishable strains of the minute virus of mice which infect mouse cells of dissimilar differentiated phenotype. Both the prototype strain and the immunosuppressive strain grow in and form plaques on monolayers of simian virus 40-transformed human fibroblasts, a finding that has allowed the comparison of several aspects of their virus-host cell interactions. Although closely related by antigenic and genomic criteria, both the prototype strain and the immunosuppressive strain are restricted for lytic growth in each other's murine host cell, that is, in T cells and fibroblasts, respectively. The host range of each virus variant appears to be specified by a genetic determinant that is stably inherited in the absence of selection. In the restrictive virus-host interaction lytic growth is limited to a small or, in some cases, undetectable subset of the host cell population, and the majority of the infected cells remain viable, continuing to grow at the normal rate without expressing viral antigens. The susceptible host cell phenotype is dominant in T lymphocyte x fibroblast fusion hybrids, implying that different cell types express different developmentally regulated virus helper functions that can only be exploited by the virus variant that carries the appropriate strain-specific determinant.

Animals↗

Induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus MHV-S.

Kirsten murine sarcoma virus-transformed, nonproducer BALB3T3 (K-BALB) cells were persistently infected with mouse hepatitis virus, MHV-S. The cultures developed plaques after infection with murine leukemia viruses. If the murine leukemia virus-infected cultures were further submitted to the UV-XC assay, comparable numbers of XC plaques were obtained. The sensitivity to murine leukemia viruses, as determined by the UV-XC assay, was higher in MHV-S-infected cells as compared to uninfected K-BALB cells.

Animals↗

Characterization of the Epstein-Barr virion-associated DNA polymerase as isolated from superinfected and drug-stimulated cells.

We reported previously that Epstein-Barr (EB) virions and detergent-treated nucleocapsids co-purified with significant amounts of DNA polymerase activity that did not resemble other known host or viral polymerases. We report here that this species of DNA polymerase activity is present at early times after infection in lymphocytes abortively lytically infected (superinfected) with EB virus. However, studies with [35S]methionine labeling suggest de novo synthesis of enzyme has not occurred. Conversely, drug-stimulated lymphocytes that synthesize EB viral late proteins and virions contain this species of polymerase to the virtual exclusion of all others. This EB viral polymerase shows a marked preference for nicked and gapped double-stranded rather than primed single-stranded DNA templates. Its processiveness as measured on primed theta X174 phage DNA template is lower than that of lymphocyte beta polymerase. The data reported here are consistent with the hypothesis that the EB virion-associated DNA polymerase is synthesized at late times in the viral life cycle as are other structural proteins but it plays an important role early after viral infection. It is known that mature herpes virion DNA (including that of EB virus) is nicked and gapped and we propose that virion polymerase repairs the viral DNA at an early stage in infection before viral DNA replication begins.

B-Lymphocytes↗

Method for phage typing group A type 49 streptococci.

A method of phage subtyping group A type 49 streptococci is described. The method is similar to that used for phage typing staphylococci, except that lysates obtained by induction with mitomycin C rather than propagated stock phages were used. Five type 49 strains were used as phage donors. Seventy-two strains of type 49 streptococci isolated from 10 worldwide sources were examined by this method. Among these strains, five distinct subtypes (I through V) could be distinguished on the basis of their lytic patterns. Only a few of the type 49 strains could not be classified into one of these phage subtypes (6% using 100 X routine test dilution). Strains from a single source were generally homogeneous with respect to their phage subtype. The method proved useful in discriminating between type 49 strains isolated from different geographical sources and from the same place in different years. Studies in progress suggest that it may be useful for subtyping other strains of special epidemiological interest, such as strains of other serological types associated with nephritis.

Bacteriophage Typing↗

LPP-1 infection of the blue-green alga Plectonema boryanum. I. Electron microscopy.

One-step growth and intracellular growth experiments were performed at high multiplicities of the virus LPP-1 during the infection of the blue-green alga Plectonema boryanum. The eclipse period lasts until 4 hr after infection, the latent period terminates at 6 hr, and the rise period continues until 14 to 16 hr after infection. The burst size was independent of multiplicity of infection over the ranges from 1 to 50. The burst size was 3,000 to 5,000 plaque-forming units (PFU) per infectious center or about 200 PFU per cell. Samples for electron microscopy were taken at characteristic times during the lytic cycle. The first sign of viral infection was the invagination of the photosynthetic lamellae at 3 hr after infection. Mature virions were visible at 4 hr. By 6 to 7 hr, many mature intracellular viral particles could be seen, with lysis beginning at 7 hr. By 10 hr after infection, all infected cells contained mature virions. No evidence for mass migration of performed viral precursors was obtained. The invagination of the lamellae could be prevented by the early addition of chloramphenicol, which implies that this process requires protein synthesis.

Centrifugation, Density Gradient↗

Antisense RNA-dependent transcription termination sites that modulate lysogenic development of satellite phage P4.

In the lysogenic state, bacteriophage P4 prevents the expression of its own replication genes, which are encoded in the left operon, through premature transcription termination. The phage factor responsible for efficient termination is a small, untranslated RNA (CI RNA), which acts as an antisense RNA and controls transcription termination by pairing with two complementary sequences (seqA and seqC) located within the leader region of the left operon. A Rho-dependent termination site, timm, was previously shown to be involved in the control of P4 replication gene expression. In the present study, by making use of phage PhiR73 as a cloning vector and of suppressor tRNAGly as a reporter gene, we characterized two additional terminators, t1 and t4. Although transcription termination at neither site requires the Rho factor, only t1 has the typical structure of a Rho-independent terminator. t1 is located between the PLE promoter and the cI gene, whereas t4 is located between cI and timm. Efficient termination at t1 requires the CI RNA and the seqA target sequence; in vitro, the CI RNA enhanced termination at t1 in the absence of any bacterial factor. A P4 mutant, in which the t1 terminator has been deleted, can still lysogenize both Rho+ and Rho- strains and exhibits increased expression of CI RNA. These data indicate that t1 and the Rho-dependent timm terminators are not essential for lysogeny. t1 is involved in CI RNA autoregulation, whereas t4 appears to be the main terminator necessary to prevent expression of the lytic genes in the lysogenic state.

Bacterial Outer Membrane Proteins↗

Role of the phi 11 phage genome in competence of Staphylococcus aureus.

Both phage ø11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ø11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ø11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (ø11) or 8325-4 (83A) at high multiplicities with phage ø11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (tsø11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ø11 of Kretschmer and Egan and tsø11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ø11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.

Azo Compounds↗

Transcriptional induction of the ubiquitin gene during herpes simplex virus infection is dependent upon the viral immediate-early protein ICP4.

Lytic infection with herpes simplex virus results in transcriptional induction of a cellular gene encoding ubiquitin, causing increased accumulation of ubiquitin RNA and protein in the infected cell. This induction, which is dependent upon viral protein synthesis, does not occur in the HSV-1 mutant tsK which is defective in the gene encoding the viral protein ICP4. Transfected cells expressing the viral ICP4 protein exhibit higher levels of ubiquitin gene transcription than untransfected controls indicating that transcriptional induction can be mediated by the ICP4 protein alone.

Animals↗

Syncytium-forming virus of common marmosets (Callithrix jacchus jacchus).

This communication describes the isolation and characterization of a new syncytium-forming virus of common marmosets (Callithrix jacchus jacchus). The virus, isolated from skin explants and peripheral leukocytes of healthy animals, induced syncytia and subsequent cytolysis of several human, simian, and rodent fibroblastic cultures and induced a carrier state in mixed fibroblastic-epithelial or epithelial cell lines. Cytoplasmic and nuclear viral antigen was demonstrated in infected cells by indirect immunofluorescence tests using serum obtained from persistently infected common marmosets. Abundant virus particles were detected within cisternae of endoplasmic reticulum of lytically infected cells by electron microscopy. The virus incorporated [3H]uridine, banded at a density of 1.14 to 1.16 g/cm3 in sucrose, and possessed ribonucleic acid-dependent deoxyribonucleic acid polymerase. No antigenic cross-reactivity was detected between the marmoset virus and simian foamy virus serotypes 1 to 8 in neutralization and immunofluorescence assays. A seroepidemiological survey of a marmoset colony revealed that 53.5% of common marmosets contained antibodies against the virus, whereas other species of marmosets maintained in the same colony remained free of antibodies.

Animals↗

Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells.

Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.

Antibodies↗

Unique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2).

OBJECTIVE: Human herpesvirus 8/Kaposi's sarcoma herpesvirus (HHV-8/KSHV) contains, in addition to genes required for viral replication, an unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that show homology to the transcription factors of the interferon regulatory factor (IRF) family. In this study we demonstrate that one of these ORFs (vIRF-2) encodes a protein with mobility of 18 kd which has distinct pattern of expression and properties from the cellular IRFs and the previously characterized vIRF-1. METHODS: We cloned vIRF-2 by polymerase chain reaction (PCR) and studied its expression by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Biologic activities were tested by chloramphenicol acetyltransferase (CAT) assay in transiently transfected mammalian cells. We characterized its DNA binding specificity by electrophoretic mobility shift analysis (EMSA) and its protein-protein interactions by in vitro pull-down assay. RESULTS: Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-positive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primary lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does not bind specifically to the oligodeoxynucleotide repeats corresponding to the interferon-stimulated response element (ISRE), but it does bind to the NF-kappa B binding site. The fusion protein generated from vIRF-2 and the RelA (p65) activation domain stimulates transcriptional activity of HIV LTR, which contains two NF-kappa B sites, but does not stimulate the interferon-beta (IFNB) promoter, which contains only one NF-kappa B site. Interaction between recombinant vIRF-2 and cellular IRFs such as IRF-1, IRF-2, and ICSBP was detected by in vitro binding assay, but no interaction between IRF-3 and vIRF-2 was found. Interaction of vIRF-2 with RelA (p65) and the carboxy-terminal part of p300 was also observed. In a transient transfection assay, vIRF-2 inhibits the IRF-1- or IRF-3-mediated transcriptional activation of interferon-alpha (IFNA) gene promoter in infected cells and downmodulates RelA (p65)-stimulated activity of HIV LTR. CONCLUSIONS: These results suggest that, by interacting with cellular transcription factors and cofactors, vIRF-2 may modulate the expression of the early inflammatory genes and potentially deregulate the immune system.

3T3 Cells↗

Endogenous TGF-beta contributes to the induction of the EBV lytic cycle in two Burkitt lymphoma cell lines.

A low proportion of cells in the BL lines P3HR-I and Akata enter spontaneously into the EBV lytic cycle, detectable by the expression of early antigens (EA). We found that both lines produce the active and inactive forms of TGF beta. It was shown earlier that a larger number of cells can be induced to enter the lytic cycle by exposing P3HR-I to phorbol esters and n-butyrate and the surface IgG-positive Akata cells to anti-IgG. We now show that the same treatments raise the level of active TGF beta release. Exposure to anti-TGF beta antibodies reduced EA induction by 75-85%. Our results indicate that induction of the viral productive cycle by the above-mentioned reagents is at least partly dependent on the activation of endogenous TGF beta production.

Burkitt Lymphoma↗

[The paradox of polyomaviruses. Lytic infection or tumor induction?].

Murine polyomaviruses can produce lytic infections in mouse cell cultures or transform in vitro rat fibroblasts through a complex interaction with key cellular regulators. After infection of newborn mice, some strains of polyomavirus induce epithelial and mesenchymal tumors. It has been described that there is a direct relationship between viral dissemination in the mouse and tumor induction. However, at a single cell level lytic infection and transformation would not be able to coexist. The existence of 3 distinct cell populations in polyoma-induced tumors, classified according to the presence or absence of viral DNA and viral capsid protein VP-1 have been described. We have reported a fourth type of cell in the neoplasms, that can express the early and the late viral genes but do not allow virus assembly, probably due to underphosphorylation of VP-1. The mechanisms of polyoma intracellular migration and dissemination in the mouse are discussed, related to the virus' ability of tumor induction.

Animals↗

Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984-1997): analysis of seroconversions to ORF65 and ORF73.

We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections.

Antibodies, Viral↗

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.

Cell Line↗

Complete genomic sequence of the lytic bacteriophage DT1 of Streptococcus thermophilus.

Streptococcus thermophilus lytic bacteriophage DT1, isolated from a mozzarella whey, was characterized at the microbiological and molecular levels. Phage DT1 had an isometric head of 60 nm and a noncontractile tail of 260 x 8 nm, two major structural proteins of 26 and 32 kDa, and a linear double-stranded DNA genome with cohesive ends at its extremities. The host range of phage DT1 was limited to 5 of the 21 S. thermophilus strains tested. Using S. thermophilus SMQ-301 as a host, phage DT1 had a burst size of 276 +/- 36 and a latent period of 25 min. The genome of phage DT1 contained 34,820 bp with a GC content of 39.1%. Forty-six open reading frames (ORFs) of more than 40 codons were found and putative functions were assigned to 20 ORFs, mostly in the late region of phage DT1. Comparative genomic analysis of DT1 with the completely sequenced S. thermophilus temperate phage O1205 revealed two large homologous regions interspersed by two heterologous segments. The homologous regions consisted of the early replication genes, the late morphogenesis genes, and the lysis cassette. The divergent segments contained the DNA packaging machinery, the major structural proteins, and remnants of a lysogeny module.

Amino Acid Sequence↗

Measles virus gene expression in subacute sclerosing panencephalitis.

RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.

Adolescent↗

Changing Epstein-Barr viral ZEBRA protein into a more powerful activator enhances its capacity to disrupt latency.

The Epstein-Barr viral transcriptional activator ZEBRA induces expression of viral early lytic genes when introduced into cells bearing latent Epstein-Barr virus. We show here that a ZEBRA-herpes simplex viral protein 16 (VP16) fusion protein induces early viral lytic gene expression in Epstein-Barr virus-containing cells more efficiently than does wild-type ZEBRA. The fusion protein is also a more powerful transcriptional activator in these cells, as assayed with reporter constructs. Our experiments also suggest that ZEBRA manifests a function required for full activity on certain natural promoters but not on promoters bearing oligomerized ZEBRA binding sites; this function cannot be provided by VP16.

Cells, Cultured↗