Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses.

The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.

Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase.

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.

Genetic recombination of human immunodeficiency virus.

We investigated genetic recombination of the human immunodeficiency virus (HIV) in a tissue culture system. A clonal cell line expressing a single integrated HIV provirus with a termination codon affecting pol gene expression was transfected with different defective mutants derived from an infectious molecular clone of HIV. Replication-competent viral particles were recovered, passaged, and plaque purified. Restriction analyses of the proviral DNA corresponding to several of these viruses indicated that their emergence was the result of genetic recombination.

Potential progenitor sequences of mink cell focus-forming (MCF) murine leukemia viruses: ecotropic, xenotropic, and MCF-related viral RNAs are detected concurrently in thymus tissues of AKR mice.

Leukemogenic mink cell focus-forming (MCF) viruses of AKR mice are believed to originate in thymic tissue via recombination between ecotropic, xenotropiclike, and endogenous MCF-related murine leukemia virus (MuLV) sequences. We have previously used a synthetic 16-base-pair MCF env-specific oligomer probe to identify subgenomic MCF-related mRNAs present in the thymus tissues of AKR mice prior to the appearance of full-length (8.4-kilobase [kb]) recombinant MCF viral RNAs (A. S. Khan, F. Laigret, and C. P. Rodi, J. Virol. 61:876-882, 1987). These potential MCF env precursors consisted of 7.2-, 3.0-, and 1.8-kb RNA species. In this study, we have determined the structure of the MCF-related mRNAs on the basis of Northern (RNA) blot hybridization analyses by using 10 different MuLV subgenomic DNA probes, determined the nucleotide sequence of a cloned cDNA segment representing the 3' portion of the 7.2-kb mRNA, and studied the expression of ecotropic and xenotropic MuLV sequences by using env-specific DNA probes. The results indicated that ecotropic, xenotropic, and MCF-related transcripts were constitutively and concurrently expressed exclusively in thymus tissue of 2-month-old AKR mice prior to detection of MCF viral RNAs. We have molecularly characterized these thymic MuLV RNAs, which may participate in formation of recombinant MCF viruses; a novel recombinant ecotropic viral RNA was identified as a putative intermediate in the stepwise generation of leukemogenic MCF MuLVs. We have also described the unique structure of the 6.0-kb MCF-related RNAs which were expressed specifically in liver and kidney tissues of AKR mice; these RNAs contained an upstream non-MuLV transcriptional regulatory element.

Amphotropic proviral envelope sequences are absent from the Mus germ line.

We derived an amphotropic murine leukemia virus (MuLV) type-specific probe for use in Southern blot hybridizations with cloned and genomic DNAs. A 133-base-pair RsaI-RsaI fragment from the 5' env region of the amphotropic viral isolate 4070A was subcloned into M13mp18 and radiolabeled in vitro. The probe detected the proviral DNAs in mink cells infected with seven different amphotropic MuLV isolates. The probe did not cross hybridize with the DNAs of molecular clones of ecotropic, mink cell focus-forming, or xenotropic MuLVs; nor did it anneal to the proviral DNAs of four xenotropic or six mink cell focus-forming viral isolates grown in mink cells. DNAs of 12 inbred laboratory mouse strains and more than 15 different wild mouse species and subspecies were examined for the presence of endogenous amphotropic env-related fragments. Amphotropic env-related sequences were found only in the DNAs of wild mice trapped in southern California in an area previously shown to harbor mice producing infectious amphotropic virus. Restriction enzyme analyses of DNAs from these mice showed that amphotropic sequences were not present as germ line copies but were the result of congenital or horizontal infection or both in this population. The DNAs of 11 various mammalian and avian species, including both natural predators of mice and squabs from the farms with virus-positive mice, lacked amphotropic envelope-related sequences.

Specific hybridization probes demonstrate fewer xenotropic than mink cell focus-forming murine leukemia virus env-related sequences in DNAs from inbred laboratory mice.

We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.

Characterization of a molecularly cloned retroviral sequence associated with Fv-4 resistance.

The murine leukemia virus (MuLV) sequence associated with the resistance allele of the Fv-4 gene (Fv-4r) was molecularly cloned from genomic DNA of uninfected mice carrying this allele. The 5.2-kilobase cloned EcoRI DNA fragment (pFv4) was shown by nucleotide sequencing to contain 3.4 kilobases of a colinear MuLV-related proviral sequence which began in the C-terminal end of the pol region and extended through the env region and the 3' long terminal repeat. Cellular sequences flanked the 3' as well as the 5' ends of the truncated MuLV sequence. Alignment of the N-terminal half of the pFv4 env sequence with ecotropic, mink cell focus-forming, and xenotropic MuLV env sequences established the relatedness of pFv4 and ecotropic MuLV env sequences. A subcloned 700-base pair segment (pFv4env) from the 5' env region of pFv4 was used as an Fv-4-specific probe; it hybridized specifically to the Fv-4r-associated proviral sequence but not to endogenous ecotropic MuLV proviral DNA under high stringency. All Fv-4-resistant mice contained the same retroviral segment associated with the same flanking cellular DNA. Expression of Fv-4r-specific mRNA was demonstrated in the spleens of Fv-4r mice but not Fv-4s mice, supporting the previously proposed resistance model based on interference.

Nucleotide sequence of a full-length human endogenous retroviral segment.

The nucleotide sequence of a full-length (8.8-kilobase) endogenous C-type human retroviral DNA (clone 4-1) is presented and compared with that of Moloney murine leukemia virus (MoMuLV) DNA. Colinearity of deduced amino acids of clone 4-1 with MoMuLV in the gag and pol regions was clearly evident, and overall amino acid homology in these regions was about 40%. Identification of the putative N terminus of gag and p30, the gag-pol junction, and the C terminus of pol could be established on the basis of sequence homology with MoMuLV. Unique characteristics of the endogenous human retroviral DNA included a tRNA Glu primer binding site separated from the 5' long terminal repeat by a pentanucleotide and a putative env sequence which does not appear to overlap the C terminus of pol and has virtually no homology with the env gene of known infectious retroviruses. Clone 4-1 represents a defective prototype of a human C-type retrovirus which integrated into the germ line some time in the distant past.

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus.

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.

Characterization and partial nucleotide sequence of endogenous type C retrovirus segments in human chromosomal DNA.

Twenty-six different murine leukemia virus (MuLV)-related clones have been isolated from a human DNA library and characterized by restriction enzyme mapping and reciprocal nucleic acid hybridization reactions. The sequence of approximately 2,600 nucleotides, spanning more than 4.0 kilobases, of one of the MuLV-related cloned human DNAs was also determined. The deduced amino acid sequence permitted the alignment of this prototype cloned human DNA segment with the p12 gag, p30 gag, p10 gag, and pol regions of Moloney MuLV. A majority of the endogenous type C retrovirus-related segments present in human DNA are approximately 6.0 kilobases in size and appear to contain a deletion of env sequences.

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.

Control of Escherichia coli growth by CO2.

Escherichia coli B dependence on CO2 for growth was demonstrated. At suboptimal CO2 concentrations the rate of growth was controlled by CO2 concentration.

Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.

Quantitative requirements for exponential growth of Alcaligenes eutrophus.

Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.

Carbon dioxide control of lag period and growth of Streptococcus sanguis.

A carbon dioxide requirement for growth of Streptococcus sanguis was readily demonstrated in a fermentor where the gas atmosphere could be controlled. Growth at a maximum rate occurred immediately in response to the appropriate CO(2) concentration; growth stopped when CO(2) was deleted. Washed inocula consisting of exponentially growing cells required a minimum of 2.4% CO(2), postexponential phase cells needed 1.2 to 1.8% CO(2) immediately and 2.4% CO(2) shortly thereafter, whereas stationary phase cells required three sequential increases in CO(2) from 0.3 to 1.8 to 2.4% within the first 90 min of growth. These CO(2) concentrations permitted each inoculum to initiate growth immediately at the same maximum rate. These results also showed that physiologically "old" cells had the same capacity for growth as "young" cells when the CO(2) concentrations were appropriate for the type of inoculum. Continued exponential growth of the culture at the same optimum rate required 2.4% CO(2). Lower concentrations of CO(2) were rate limiting and the resulting exponential rate was proportional to the CO(2) concentration. The "normal" lag period of S. sanguis appears to be an artifact induced by a CO(2) deficiency.

Bicarbonate requirement for elimination of the lag period of Hydrogenomonas eutropha.

Carbon dioxide and oxygen concentrations have a profound effect on the lag period of chemoautotrophically grown Hydrogenomonas eutropha. Minimum lag periods and high growth rates were obtained in shaken flask cultures with a prepared gas mixture containing 70% H(2), 20% O(2), and 10% CO(2). However, excessively long lag periods resulted when the same gas mixture was sparged through the culture. The lag period was shortened in sparged cultures by decreasing both the pO(2) and the pCO(2), indicating that gas medium equilibration had not occurred in shaken cultures. The lag period was completely eliminated at certain concentrations of O(2) and CO(2). The optimum pO(2) was 0.05 atm, but the optimum pCO(2) varied according to the pH of the medium and physiological age of the inoculum. At pH 6.4, the pCO(2) required to obtain immediate growth of exponential, postexponential, and stationary phase inocula at equal specific rates was 0.02, 0.05, and 0.16 atm, respectively. With each 0.3-unit increase in the pH of the medium, a 50% decrease in the CO(2) concentration was needed to permit growth to occur at the same rate. The pCO(2) changes required to compensate for the pH changes of the medium had the net effect of maintaining a constant bicarbonate ion concentration. Initial growth of H. eutropha was therefore indirectly related to pCO(2) and directly dependent upon a constant bicarbonate ion concentration.