Host Range Mutants Research Articles

Propagation and in vitro studies of previously non-cultivable enteral adenoviruses in 293 cells.

Enteral adenovirus (EAd) isolates which characteristically fail to propagate in several human cell lines grow readily in 293 cells, an adenovirus type 5 transformed line. EAd infection of 293 cells is associated with high levels of 31S DNA and late viral protein synthesis, the appearance of a progressive adenovirus-like cytopathic effect, and the efficient assembly of progeny virions. The DNA of an EAd strain grown in 293 cells possesses restriction endonuclease profiles similar to those reported for EADs recovered directly from stools. In their ability to replicate efficiently in 293 cells but not in other human cell lines. EAds appear to behave like adenovirus host range mutants.

Infection of Escherichia coli K-12 by bacteriophage φX-174

By several different biochemical tests, a previously described isolation procedure was shown to produce host-range mutants of φX-174 capable of infecting some strains of Escherichia coli K-12. One of these mutants (φXK-9) has a wide host range which includes E. coli C, B, and Shigella Y6R. By fragment mapping the site of the host-range mutation was shown to be in gene F.

Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

Survey of phytopathogenic pseudomonads for a restriction and modification system active on the double-stranded ribonucleic acid phage ϕ6

A total of 380 pseudomonad strains from 39 nomenspecies and 41 strains from 7 other bacterial genera were screened for a double-stranded ribonucleic acid modification and restriction system using the double-stranded ribonucleic acid bacteriophage ϕ6. Of these 421 strains, 8 showed the low plating efficiency (10−5 to 10−7) characteristic of such a system. Howver, the phage propagated in 7 of the 8 were host-range mutants; the remaining strain showed some characteristics of a host-modification system but the results were equivocal.

Host range deletion mutant of vaccinia virus defective in human cells

A vaccinia virus host range (hr) mutant unable to multiply in most human cell lines assayed has been isolated after nitrous acid mutagenesis. This mutant also displayed various alterations in plaque morphology and cytopathic effect on permissive cell lines. The block in multiplication in human cells was at an early stage of infection. Only early cytoplasmic RNA and early viral-induced polypeptides could be detected and there was no evidence of morphological events within viroplasms. Protein synthesis constantly declined as infection proceeded suggesting either that early viral mRNA was unstable or that the mutant virus was defective in a step necessary for the maintenance of translation. Restriction enzyme digestion of DNA from the hr mutant revealed that it was deleted of about 12.6 × 10 6 daltons in the left-hand end of the genome leaving intact a fragment containing the terminal crosslink. In both permissive and nonpermissive cells the mutant failed to induce the synthesis of an early 42K polypeptide which could thus be encoded within a region of the deleted sequence. The failure to segregate the various phenotypic and biochemical properties of the hr mutant from one another through recombination with a temperature sensitive mutant indicated that the pleiotropic characteristics of the mutant virus were due to the deletion.

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.

Evidence for functional relatedness of products encoded by the transforming sequences of human adenovirus types 5 and 12

Host-range and host-range deletion mutants of type 5 adenovirus belonging to two complementation groups, whose mutational lesions map physically within the left 11% of the type 5 genome, were efficiently complemented in mixed infection by type 12 adenovirus. The complementation of these early function mutants by type 12 is in contrast to the lack of complementation found previously for two other groups of early function mutants, and it is consistent with functional relatedness between products encoded by early region 1 of the respective serotypes. No evidence for recombination between the type 5 mutants and type 12 was found.

Locations of adenovirus genes required for the replication of adenovirus-associated virus.

We have used DNA transfection to identify several regions of the adenovirus genome needed to induce replication of the defective parvovirus, adenovirus-associated virus (AAV). Previous studies have indicated that only early adenovirus functions are needed to aid the replication of AAV. In this report, we demonstrate that three restriction endonuclease fragments of adenovirus DNA are necessary for production of infectious AAV in 293-31 cells (an adenovirus type 5-transformed human embryonic kidney cell line). These fragments map from 28.5 to 29.4, 59.5 to 75.9, and 89.7 to 100 map units on the adenovirus type 2 genome and correspond to the locations of the VAI RNA gene, early region 2, and early region 4, respectively. The 293-31 cell line, which has been found to express early region 1A and 1B proteins, alone is incapable of supporting AAV replication or even AAV DNA synthesis. Additional experiments with adenovirus type 5 host range mutants (group I, hr1 and group II, hr7) indicate, however, that early region 1A provides an essential function(s) for AAV replication, whereas early region 1B probably does not.

Isolation and characterization of polyoma host range mutants that replicate in nullipotential embryonal carcinoma cells.

Polyoma wild-type virus replicates in most murine differentiated cells but fails to produce virus in murine embryonal carcinoma cells. Polyoma host range mutants have been isolated that replicate in several nullipotential embryonal carcinoma cell lines but fail to replicate in a pluripotential cell line. The final virus yield of these host range mutants is dependent upon the multiplicity of infection in both differentiated cells and nullipotent embryonal carcinoma cells. Two independently derived host range mutants contain the same single base pair change (A.T to G.C) and a 33- and 67-base pair duplication of those viral DNA sequences containing this point mutation. This duplication of viral DNA is located at 69 map units on the polyoma genome on the late gene side of the origin of viral DNA replication (70.5 map units). This type of mutation suggests several models to explain the polyoma host range restriction in embryonal carcinoma cells.

OmpC and LamB proteins can serve as substitute receptors for host range mutants of coliphage TuIa.

Coliphage TuIa, which uses the OmpF protein as a receptor, can adapt by mutation to using instead the OmpC or LamB protein, or both. Most of the phage mutants retained the ability to use the OmpF protein, when present, but one class of mutants lost this ability and could only use the OmpC protein.

The white pock mutants of rabbit poxvirus II. The early white pock (μ) host range (hr) mutants of rabbit poxvirus uncouple transcription and translation in nonpermissive cells

A biochemical examination of the white pock (μ) host range (hr) mutants of rabbit poxvirus (RP μhr mutants) on the prototypic nonpermissive pig kidney (PK) cell line allows them to be divided into two groups, “early” or “late,” based on their ability to synthesize viral DNA in these cells. We have studied two early mutants (those unable to synthesize viral DNA) and found that by 8–10 hr after infection of PK cells all translation ceases; however, viral RNA synthesis continues unabated. These data provide evidence for the active control of translation during poxvirus infection. Even though the two mutants both inhibit translation under nonpermissive conditions, they clearly differ biochemically from one another in their total transcriptional capacity. An exmaination of the RNA made by these two RP μhr mutants under nonpermissive conditions further reveals that at times, which would be equivalent to “late” in a normal infection, one mutant has transcribed only 62% and the other 84% of the RNA sequences synthesized in a wild-type infection. Furthermore, transcription continues long after translation has stopped. Both mutants have also been found to be more refractile to uncoating in nonpermissive cells than wild-type virus. We find that the rate of the host-dependent formation of subviral cores is impaired as is the subsequent step of viral-dependent uncoating of the cores. A defective uncoating process might be related to the reduced complexity of transcription observed with these mutants. Despite their similarities, our results suggest that the two mutants are interrupted at different stages within the early phase of infection and that both poxvirus transcription and translation are actively regulated prior to the onset of viral DNA synthesis.

A temperature-dependent host range mutant of fowl plague virus (FPV)

Temperature-dependent (td) host range mutants of the mammalian cell adapted Dobson strain of fowl plague virus (FPV-B) have been isolated after mutagenization by 5-fluorouracil. These mutants form plaques on chick embryo cells (CEC) at 34 and 39.5° but on L cells only at 34°. The original FPV-B grows equally well at 34 and 39.5° on both types of cells. The is defect was located in the gene coding for the P 1 protein by genetic analysis of various recombinants between one of these mutants (td4) and the Rostock strain of FPV tested for their ability to form plaques on L cells. Analysis of viral polypeptides and RNAs synthesized in L cells by the td4 mutant at 34 and 39.5° indicated that the functional defect is at the level of RNA replication. This suggests an involvement of a host cell factor in viral RNA replication, which in CEC helps the P 1 protein to overcome its heat sensitivity.

Physical location of host-range mutations of adenovirus type 5; deletion and marker-rescue mapping

Sites of host-range mutations have been mapped physically on the adenoviral genome by both recombinational mapping with host-range deletion mutants and by marker rescue. The results from the two methods are in excellent agreement; one group of mutations is assigned to the sequence between 1.3 and 3.7 units and the other set to the sequence between 6.1 and 8.5 units.

Adenovirus early region 1b gene function required for rescue of latent adeno-associated virus

Various early and late temperature-sensitive mutants of adenovirus (Ad) were able to support adeno-associated virus (AAV) DNA replication in either coinfections or in the rescue of AAV from latently infected human (Detroit 6) cells. Group I Ad host range mutants ( hr 3, early region 1a) were unable to help AAV DNA replication in either case. Group II Ad host range mutants ( hr 6, early region 1b), however, were able to help AAV DNA replication in coinfection but were unable to rescue AAV DNA replication in latently infected Detroit 6 cells. Thus, an Ad function(s), in addition to those required for AAV DNA replication, is needed for AAV rescue.

Correlation between the expression of an Escherichia coli cell surface protein and the ability of the protein to bind to lipopolysaccharide.

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying "foreign" genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA(+) phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.

Early viral proteins in HeLa cells infected with adenovirus type 5 host range mutants

Several classes of adenovirus type 5 host range mutants have been isolated, These mutants replicate in Ads-transformed human embryonic kidney cells (293 cells) but at low multiplicities of infection fail to replicate in HeLa cells. Experiments are presented that demonstrate that these host range mutants form two complementation group;: group I maps in region E1a of the adenovirus genome and contains mutants hr1, hr2, dl311, and dl312; group II maps in region Elb and includes mutants hr6, hr7, dl313, and sub315. Mutants dl314 and sub316 both fail to efficiently complement any of the group I and II host range mutants. At low multiplicities of infection most group I mutants failed to produce detectable levels of virus proteins derived from regions E1, E2, or E3 of the viral genome. This group I phenotype was markedly depen(lent upon the multiplicity of infection; at a multiplicity of 100 PFU/cell, hr1-infected HeLa cells contained region E1b, E2, and E3 proteins, Group II mutants failed to produce only region E1b proteins; some of these mutants overproduced (two- to fivefold) region E2 and E3 proteins,

The white pock mutants of rabbit poxvirus I. Spontaneous host range mutants contian deletions

Restriction enzyme analysis of five different spontaneously generated white pock (μ), host range (hr) mutants of rabbit poxvirus (RPμhr mutants) reveals a series of overlapping mutations which can be partially explained by deletion. Although each of the mutants has deleted some genetic material, one mutant has a larger altered region of DNA than can be accounted for by simple deletion. The deletions originate in the far left-hand region of the DNA near or within the inverted terminal repeat. For at least three of the mutants, the deletion extends into the terminal repeat region. The deletions range in size from 3 × 10 6 to 20 × 10 6 daltons and affect mostly unique viral sequences. Thus, up to 17% of the viral DNA can be eliminated without impairment of growth of the resulting virus on certain cells. The observed lack of reversion and the failure to detect either wild-type recombinants or complementation in mixed infection between pairs of mutants are biological properties expected for a family of mutants that genetically represent a series of partially overlapping deletions.

Host-dependent modification of bacterial virus T3 affecting its adsorption ability

The adsorption and thus the growth of T3 on Escherichia coli W cells ( E. coli K12 derivative) depends decisively on the host strain on which the virus was previously propagated. Depending on the modification conferred to the virus by its last host, its efficiency of plating on E. coli W varies between 10 −7 and 10 −1. This does not reflect the appearance of T3 host-range mutants, but a fully reversible modification of genotypically unchanged T3 wild-type phage.

Sub-classification of phage types of Salmonella weltevreden and typing of strains by an extended phage typing scheme.

By the use of two adapted phage preparations of Typing phage II the S. weltevreden phage types 4 and 5 could be classified into two sub-types each and phage types 9 and 10 into three sub-types each. the 1094 strains of S. weltevreden could be classified into a total of sixteen phage types including the sub-types. The host range mutants of Typing phage II were distinct from the parent strain. After adaptation to two insensitive strains, one of the new preparations, phi IIA lost its affinity to some strains which were lysed by the parent phage strain but gained lytic affinity for a few others that were originally insensitive. The second preparation phi IIB showed an increase in lytic range as expected. Antigenically these preparations were shown to be related but not identical. The possible reasons for serological non-identity of host range mutants with the parent strain have been discussed.