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Here, we describe the evolution of antigenic escape variants in a rhesus macaque that developed unusually high neutralizing antibody titers to SIVmac239. By 42 weeks postinfection, 50% neutralization of SIVmac239 was achieved with plasma dilutions of 1:1,000. Testing of purified immunoglobulin confirmed that the neutralizing activity was antibody mediated. Despite the potency of the neutralizing antibody response, the animal displayed a typical viral load profile and progressed to terminal AIDS with a normal time course. Viral envelope sequences from week 16 and week 42 plasma contained an excess of nonsynonymous substitutions, predominantly in V1 and V4, including individual sites with ratios of nonsynonymous to synonymous substitution rates (dN/dS) highly suggestive of strong positive selection. Recombinant viruses encoding envelope sequences isolated from these time points remained resistant to neutralization by all longitudinal plasma samples, revealing the failure of the animal to mount secondary responses to the escaped variants. Substitutions at two sites with significant dN/dS values, one in V1 and one in V4, were independently sufficient to confer nearly complete resistance to neutralization. Substitutions at three additional sites, one in V4 and two in gp41, conferred moderate to high levels of resistance when tested individually. All the amino acid changes leading to escape resulted from single nucleotide substitutions. The observation that antigenic escape resulted from individual, single amino acid replacements at sites well separated in current structural models of Env indicates that the virus can utilize multiple independent pathways to rapidly achieve similar levels of resistance.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.

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.

Extracellular murine leukemia virus (MLV) reverse transcriptase activity was decreased by interferon treatment in four interferon-sensitive mouse cell lines which were chronic MLV producers. In three cell lines which were relatively insensitive to interferon, extracellular enzyme activity remained unchanged by interferon treatment. The concentrations of interferon used had no effect on DNA synthesis or cell replication of AKR,C+ cells which were chronic producers of AKR-MLV. In AKR,C+ cultures interferon treatment also had no effect on the level of intracellular viral reverse transcriptase activity in spite of an inhibition of extracellular enzyme activity. Treatment of AKRC+ cultures with interferon for 9 days inhibited extracellular viral reverse transcriptase levels throughout the period of treatment; however, the intracellular enzyme activity remained unchanged, and concentrations of viral p30 (gs) antigen were increased in the interferon-treated cells. When the cells were washed to remove interferon, however, virus production rapidly rose and intracellular p30 antigen fell to the levels of untreated AKR,C+ cells. These and previously reported results suggested that in interferon-treated AKR,C+ cells virus production is inhibited at a late step in the MLV replication cycle, either directly or through the inhibition of the production of a protein required for virus assembly.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.

Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.