Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA.

The M78 protein of murine cytomegalovirus exhibits sequence features of a G protein-coupled receptor. It is synthesized with early kinetics, it becomes partially colocalized with Golgi markers, and it is incorporated into viral particles. We have constructed a viral substitution mutant, SMsubM78, which lacks most of the M78 ORF. The mutant produces a reduced yield in cultured 10.1 fibroblast and IC21 macrophage cell lines. The defect is multiplicity dependent and greater in the macrophage cell line. Consistent with its growth defect in cultured cells, the mutant exhibits reduced pathogenicity in mice, generating less infectious progeny than wild-type virus in all organs assayed. SMsubM78 fails to efficiently activate accumulation of the viral m123 immediate-early mRNA in infected macrophages. M78 facilitates the accumulation of the immediate-early mRNA in cycloheximide-treated cells, arguing that it acts in the absence of de novo protein synthesis. We conclude that the M78 G protein-coupled receptor homologue is delivered to cells as a constituent of the virion, and it acts to facilitate the accumulation of immediate-early mRNA.

UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells.

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.

Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.

Viruses and apoptosis.

Apoptosis is an active process of cell death that serves diverse functions in multicellular organisms, and under physiological conditions, it is tightly controlled. Many virus genomes encode gene products that modulate apoptosis, either positively or negatively, and induction of apoptosis often contributes directly to the cytopathogenic effects of the viruses. Inhibition of apoptosis by viruses, on the other hand, may prevent premature death of infected cells, thereby facilitating viral replication, spread, or persistence.

Negative regulation of the BZLF1 promoter of Epstein-Barr virus.

The Epstein-Barr virus BZLF1 gene product (ZEBRA) is a transcriptional activator whose expression in latently infected B cells is sufficient to induce the viral lytic cycle. Since there is no transcription of BZLF1 during latency, we carried out experiments to determine whether cis-acting negative elements in the BZLF1 promoter contribute to the lack of expression during this phase of the virus cycle. A series of deletion plasmids encompassing positions -551 to +14 of the BZLF1 promoter region were constructed and tested for the ability to drive chloramphenicol acetyltransferase (CAT) gene expression in the absence of inducing agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and anti-immunoglobulin. Expression from the intact 551-bp region was very weak in most of the cell lines tested, but deletion of 165 bp from the 5' end caused a sevenfold increase in expression of CAT. Within these 165 bp, a minimal 48-bp region was sufficient to down regulate the expression of a simian virus 40/CAT fusion plasmid. The 48-bp negative element consists of 7-bp dyad symmetry elements separated by 27 bp. The rightmost half of the dyad symmetry element partially overlaps a region which has a 14-of-15-bp homology to the human cytoskeletal gamma-actin promoter.

Adenovirus tumor-specific transplantation antigen is a function of the E1A early region.

Viable recombinant adenoviruses that carry a portion of the type 12 E1A and E1B transcription units in a type 5 background were used to identify genes controlling expression of the adenovirus tumor-specific transplantation antigen (TSTA). The TSTA immunity is not crossreacting between the group A and group C adenovirus serotypes. Viruses carrying the E1A region (sub370-12E1A), or both E1A and E1B (sub370-12E1AB) regions of Ad12, induce a strong transplantation immunity against tumors induced by syngeneic cells transformed with adenovirus 12, but fail to induce any protection against syngeneic cells transformed with adenovirus 2. Immunization with the virus carrying only the E1B region (sub370-12E1B) of adenovirus 12 induces no immunity to adenovirus 12 transformed cell line, but confers a strong protection against cells transformed with adenovirus 2. These results provide strong evidence that the adenovirus tumor-specific transplantation antigen is a function of the E1A early region.

Tumorigenicity of adenovirus-transformed cells: region E1A of adenovirus 12 confers resistance to natural killer cells.

Sensitivity of a library of rat cells transformed in vitro with viable recombinant adenoviruses to natural killer (NK) cells and allogeneic cytotoxic T cells has been studied and correlated with their oncogenic potential in syngeneic rats. All cell lines transformed with the sub370-12E1AB virus (containing E1A and E1B regions of Ad12) and with the sub370-12E1A virions (containing the E1A region of Ad12 and the E1B region of Ad5) showed a high degree of resistance to NK cells. The cell lines transformed with the sub370-12E1B virus (containing the E1A region of Ad5 and the E1B region of Ad12) were highly sensitive to NK cytotoxicity. While all cell lines transformed with virions containing the E1A genes of Ad5 expressed high levels of class I MHC antigen, only three of eight cell lines containing the E1A region of Ad12 showed detectable levels by flow cytometric analysis after staining with specific antibodies. All cell lines containing E1A genes of Ad5 were killed by in vitro generated allogeneic cytolytic T cells. Only three of eight cell lines containing the E1A region of Ad12 were killed by such CTLs; the level of cytotoxicity, however, did not reach that seen with the cells containing the E1A genes of Ad5. All cell lines containing the E1A and E1B genes of Ad12 were highly tumorigenic. Only two of four cell lines transformed with virus containing the E1A genes of Ad12 and E1B region of Ad5 were tumorigenic. The efficiency of tumor induction was low and the latent period was long confirming the importance of the E1B region. None of the cell lines transformed with virus containing the E1A region of Ad5 and the E1B genes of Ad12 were tumorigenic, reflecting their high degree of sensitivity to both natural and induced cellular immunity. Expression of the E1A region of Ad12 in transformed cells modulates not only the level of class I MHC antigens, but also confers resistance to NK cell cytotoxicity.

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)

The significance and nature of defective interfering viruses.

Deletions in viral genomes appear to be a common occurrence in the replication of all DNA and RNA viruses which have been adequately studied. Such defective genomes can replicate in the presence in the same cell of a helper virus as long as the deletion does not involve the initiation site for genome replication. Coinfection of a cell with defective and "normal" infectious virus leads to reduction in the yield of the latter. The nature of DI viruses and genomes found in Sindbis virus-infected vertebrate cells during "undiluted passage" series is discussed. This procedure leads to the accumulation of progressively shorter viral RNA genomes with internal deletions. The enrichment is limited to genome lengths which are integral fractions (1/2, 1/3, 1/4, etc.) of the complete genome, and these are also found in viral particles released at the corresponding passage levels. It is believed that the selective accumulation of these fragments is governed by constraints of assembly which demand that one full genome equivalent be packaged in a released particle. In contrast to vertebrate cells, cultured mosquito cells do not seem to produce or "recognize" DI particles. Possible implications for the epidemiology of arthropod-transmitted alphaviruses are presented.

Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.

Isolation and propagation of a segment of the simian virus 40 genome containing the origin of DNA replication.

Heteroduplex DNA molecules formed from two DNAs that differ from each other by a deletion can be cleaved at the mismatched region (a deletion loop) with the single-strand-specific S1 endonuclease. A heteroduplex DNA molecule, constructed from the DNA of simian virus 40 (SV40) mutant with a deletion of the map region 0.54-0.55 and the DNA of a second SV40 mutant having a deletion of the map segment 0.70-0.73, is cleaved twice with S1 endonuclease. One of the products is a DNA fragment of about 0.13 the length of SV40 DNA which contains the origin of SV40 DNA replication (0.67 on the SV40 DNA map).

Biochemical procedure for production of small deletions in simian virus 40 DNA.

A simple biochemical procedure for producing small deletions (15 to 50 base pairs) at virtually any location in simian virus 40 DNA has been developed. The steps involved are: cleavage of the closed-circular DNA to produce a linear structure followed by 5'-exonuclease digestion to expose a short single-stranded segment at each 3' end of the molecule. Mutants containing deletions at the site of the cleavage are obtained by infecting permissive monkey kidney cells with the exonuclease-treated DNA in the presence or absence of a helper DNA (depending upon whether or not the site of cleavage and therefore the deletion occurred in a gene required for vegetative multiplication). In this paper viable mutants with deletions at the HpaII endonuclease cleavage site (0.735 map position) and defective trans-complementable mutants with deletions at the EcoRI endonuclease cleavage site (0/1.0 map position) were isolated.

Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in simian virus 40.

S1 nuclease (EC 3.1.4.X), a single-strand-specific nuclease, can be used to accurately map the location of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a mixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and reannealed to form heteroduplexes. S1 nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined.

Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus.

Cultures of Aedes albopictus cells persistently infected with wild-type Sindbis virus (SV-W) give rise to small plaque-forming mutants which are also temperature sensitive. These mutants, designated SV-C, are neutralized by antiserum produced against SV-W. Mutant ts clones were isolated from SV-C by plaque purification. After serial undiluted passage in BHK or mosquito cells, each of the clones gave rise to ts(+) revertants which, however, remained mutant with respect to plaque morphology. Nineteen of 20 clones derived from SV-C were RNA(+), and one was RNA(-) (SV-C-2). The RNA synthesizing activity, once induced in infected cells by SV-C-2, was stable at the nonpermissive temperature (39.5 C). All clones derived from SV-C were inactivated at 60 C much more quickly than was SV-W. It was not possible to demonstrate complementation between any of the SV-C clones.

Homologous viral interference in Aedes albopictus cultures chronically infected with Sindbis virus.

Complete homologous interference is demonstrated in cultures of Aedes albopictus cells chronically infected with Sindbis virus. The interference occurred before there was any detectable RNA synthesis by the superinfecting virus.