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Reverse transcription of the retroviral RNA genome into proviral DNA is a hallmark of retroviruses. Upon infection, the process of reverse transcription is initiated near the 5' end of the genomic RNA molecule where the 3' 18 nucleotides of a tRNA molecule derived from the former host cell are annealed to the primer binding site (PBS) sequence. The PBS sequence mediates two functions of vital importance during reverse transcription: it serves as a tRNA binding site and hence facilitates initiation of reverse transcription, and through base paring with a DNA copy of the 18 3' nucleotides of the tRNA molecule generated during plus-strand DNA synthesis, it mediates the second template switch of reverse transcription (14). The tRNA primer molecule is derived from the host cell tRNA population, and different viruses utilize different tRNA molecules as primers for reverse transcription. Murine leukemia viruses (MLV) (36) and human T-cell leukemia virus (41) replicate via a tRNA^Pro, human immunodeficiency virus (HIV) (44) and mouse mammary tumor virus (27) replicate via a tRNA₃^Lys, while avian retroviruses (38) replicate via a tRNA^Trp primer molecule. Within a given virus species the sequence of the PBS, and thus the utilization of a specific tRNA isoacceptor, is highly conserved partly due to the inherent conservative mechanism of reverse transcription.

Molecular adaptation of a given retrovirus towards a single tRNA species is likely to be favorable by increasing the rate of correct initiations of reverse transcription and the fidelity of the second template switch. Genetic experiments with PBS-modified HIV-1 and avian viruses in cell culture have confirmed preferential tRNA usage in both viruses (11, 21, 45, 47). Likewise, a number of biochemical studies have demonstrated the presence of specific interactions between mature HIV-1 reverse transcriptase (RT) and tRNA₃^Lys, presumably through RT recognition of the anticodon loop (4, 5, 8, 30, 37). For the avian viruses, tRNA^Trp recognition by the avian RT dimer has been shown biochemically (3, 5, 15) and base pairings between tRNA^Trp and the viral genome upstream from the PBS sequence have been demonstrated in genetic studies of mutant viruses (1, 9). In contrast to HIV and the avian retroviruses, MLV may be less stringent in its use of a tRNA replication primer. Accordingly, we have previously shown that retroviral vectors derived from Akv-MLV may be efficiently transduced with either tRNA_1,2^Gln or tRNA₃^Lys (24) or with a genetically engineered tRNA-like primer molecule (25). Previous biochemical investigations have not revealed evidence of tRNA primer preferences in MLV (5, 33). Furthermore, analysis of pol mutants of Moloney MLV demonstrated that the presence in MLV virions of tRNA^Pro correctly annealed to the PBS is independent of products of the pol gene (13). Additional indications for a less stringent use of tRNA primers in MLV come from sequencing of MLV-related viruses endogenous to the murine genome carrying PBSs that match tRNA^Gln (10, 31).

Aside from having pivotal functions during reverse transcription, the PBS sequence that matches tRNA^Pro has been found to overlap with a negatively acting cis element that mediates provirus transcriptional silencing in the mouse germ line and in undifferentiated embryonal cell lines (6, 17, 46). Interestingly, it was recently demonstrated that the negatively acting cis element overlapping the proline PBS may affect the transcriptional activity of retroviral vectors in several lymphoid and myeloid cell types (7). Conceivably, these types of effects of PBS alterations on virus-host interactions might be revealed in mouse models of MLV pathogenesis.

As a first part of such studies, we chose to study the replication and pathogenicity of PBS-modified mutants of the rapidly disease-inducing virus SL3-3, altered to replicate via tRNA₁^Gln, tRNA₃^Lys, or tRNA_1,2^Arg. The aim of this study was dual: first, to analyze whether PBS-modified viruses can replicate stably and efficiently in vivo with tRNA primers other than tRNA^Pro and, second, to investigate whether the introduced PBS modifications overlapping the negatively acting cis element influence the pathogenicity of the viruses. SL3-3 MLV is a highly lymphomagenic, nonacutely transforming virus that induces T-cell lymphomas with 100% incidence (35). Depending on the mouse strain, the latency period varies from 2 to 6 months (12, 18). The T-cell lymphomagenic potential of SL3-3 has been mapped to the U3 region of the long terminal repeat (LTR) (18, 20) and is likely mediated primarily through binding of T-cell-specific transcription factors, resulting in a high rate of replication and rendering the virus a strong insertional mutagen in the T-cell compartment of the hematopoietic system.

The viruses were derived from a pBR328 subclone of SL3-95 (19), T464, containing a functional SL3-3 provirus surrounded by sequences of genomic DNA. A deletion mutant, T464(PvuI), lacking the 5' LTR region and part of the leader region was linearized with SpeI and ligated to a 735-bp EcoRI-SpeI fragment derived from either pPBS-gln1, pPBS-lys3, or pPBS-arg1,2 and encompassing the 5' LTR and 157 bp of the leader region of Akv-MLV. Three SL3-3 replication-competent viruses with mutations in their PBS sequences at Gln1, Lys3, Arg1,2, hereinafter referred to as SL3-3-Gln1, SL3-3-Lys3, and SL3-3-Arg1,2, were thereby created. pPBS-gln1 and pPBS-lys3 have previously been described (24), and pPBS-arg1,2 is syngeneic to these vectors except in the PBS sequence. Within the leader region SL3-3 differs from Akv-MLV by having a cytosine at position 163 and adenosine residues at positions 235, 247, and 249. SL3-3 also differs from Akv by having a 2-bp guanosine insertion after position 246. The effect of introducing these leader mutations was analyzed through construction of an additional mutant, SL3-3-Pro/Akv UTR, which contains the wild-type (wt) proline-specific PBS and the Akv-specific leader. The viruses were generated by direct transfections of purified ligation reaction mixtures (total amount of DNA, approximately 5 µg) into NIH 3T3 cells by calcium phosphate precipitation, and the emergence of viruses was monitored by assaying the supernatant for RT activity (22). For the generation of wt SL3-3, T464 containing the proviral clone of SL3-3 was transfected into NIH 3T3 cells and is hereafter referred to as SL3-3-Pro. To verify the presence of the PBS mutations, RNA was purified from pelleted virus particles and subjected to RT-PCR followed by direct automated sequencing of a 200-bp region encompassing the PBS. Randomly primed reverse transcription was performed with a First Strand cDNA synthesis kit (Pharmacia), and PCR amplification was done with oligonucleotides matching regions within SL3-3 U3 (primer 1, 5'-TCCGAATCGTGGTCTCGCTGATCCTTGG-3', matching SL3-3 nucleotide positions 69 to 96) and gag (primer 2, 5'-TAGGGTCAGACTCAGAGGGGTGGT-3', specific for upstream MLV gag sequences and matching Akv positions 677 to 654). Sequencing was performed with primer 3 (5'-CGCAGGCGCAAAAAGTAGATGC-3'), which is specific for Akv positions 268 to 289. All PCRs in this study were performed in 100 µl of PCR buffer (Perkin-Elmer) containing 25 pmol of each primer, 0.2 mM each deoxynucleoside triphosphate, and 3 U of AmpliTaq Gold polymerase (Perkin-Elmer). All viruses were found to replicate stably in cell culture regardless of their PBS sequence (data not shown). To evaluate the amount of infectious particles produced, infectious-center assays were performed with a rabbit-anti-p30 antibody as described previously (40). The viral titers in the supernatants of the NIH 3T3 producer cell lines were determined in several independent assays to be 1 × 10⁶ to 1 × 10⁷, 0.4 × 10⁶ to 0.5 × 10⁶, and 1 × 10⁶ to 8 × 10⁶ infectious particles per ml of supernatant for SL3-3-Gln1, SL3-3-Lys3, and SL3-3-Arg1,2, respectively, and thus to be within a 1.5-log-unit range of that of wild-type SL3-3-Pro, which was 0.8 × 10⁶ to 1 × 10⁶ infectious particles per ml of supernatant.

To investigate the stability of the introduced PBS mutations in an in vivo model and to test their possible effects upon tumorigenesis, 100-µl aliquots of medium containing infectious particles were injected intraperitoneally into newborn inbred NMRI mice. The mice were checked for tumor development on 5 days of the week, and the mice were killed at the time of apparent illness or tumor development. Tumors were diagnosed on the basis of gross appearance of lymphoid organs as previously described (39) and according to cytologic and anatomic criteria (34). To monitor the development of viremia in the mice 2 weeks postinfection, blood samples (2.5 µl) drawn from the tail vein were analyzed for the presence of infectious particles by infectious-center assays (40). All mice injected with wt SL3-3-Pro and SL3-3-Gln1 carried infectious virus particles in peripheral blood. In contrast, 13 of 14 mice injected with SL3-3-Arg1,2 and 4 of 14 mice injected with SL3-3-Lys3 were found to be viremic at day 14 postinfection at the level of sensitivity of this assay. All viruses tested in this study were highly tumorigenic, giving rise to lymphomas in all of the injected mice (Fig. 1A and Table 1). All viruses induced tumors within a narrow time window of around 60 days, with the exception of one mouse injected with SL3-3-Lys3 and one mouse injected with SL3-3-Arg1,2, in which the tumors were detected at 142 and 119 days postinfection, respectively. Interestingly, lymphoma inductions by the PBS-modified viruses were significantly delayed compared to that of the wt virus (log rank test, P < 0.05 for SL3-3-Gln1 and SL3-3-Arg1,2 and P < 0.001 for SL3-3-Lys3). Furthermore, tumor induction by the SL3-3-Lys3 mutant was significantly delayed compared to that of the SL3-3-Gln1 mutant (log rank test, P < 0.05). To analyze whether the Akv-MLV-specific alterations introduced in the 5' leader region affected the time course of tumor induction, we performed a separate series of animal experiments in which the tumor induction of the SL3-3-Pro/Akv UTR mutant was compared to that of wt SL3-3 (Fig. 1B and Table 1). Since no significant differences in disease induction were found among these viruses, we conclude that the delays in tumor induction observed for the PBS-modified viruses are attributable mainly to the PBS alterations. However, a minor role for the Akv-MLV-specific alterations in the 5' UTR cannot be excluded.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Lymphoma development in inbred NMRI mice injected with wt and mutated SL3-3 MLVs. The cumulative mortality is plotted as a function of time after virus injection. (A) PBS-modified SL3-3 viruses; (B) 5' UTR-modified virus.

To analyze at the molecular level the disease tropism of the mutant viruses, Southern blotting was performed. Genomic DNA purified from tumors was digested with HindIII, blotted, and probed for the presence of genomic rearrangements within the T-cell receptor (TCR) -chain with two probes specific for the TCR-joining regions (J1 and J2) (2). A representative autoradiogram is shown in Fig. 2. With a few exceptions where the blotting results were inconclusive, the tumors harbored genomic rearrangements within this region and hence represented predominantly T-cell lymphomas (Table 1). We subsequently tested for rearrangements within the immunoglobulin (Ig)  light chain (23) and found no genomic rearrangements in any of the analyzed tumors, consistent with the pathological finding of enlarged thymuses (Table 1). Hence, we conclude that the PBS-modified SL3-3 viruses, like wt SL3-3, induce T-cell lymphomas. The number of integrated ecotropic viruses per tumor was investigated with a probe specific for the ecotropic env gene (23). The provirus copy numbers varied between 1 and 7, with no significant mutant-dependent differences (data not shown). The c-myc gene has previously been reported to be frequently involved in T-cell lymphomas induced by SL3-3, with 20 to 25% of tumors showing genomic rearrangements as a result of proviral integration into the c-myc promoter region (16, 43). With a probe specific for the c-myc promoter (43), clonal rearrangements within the c-myc promoter were detected in 25% (13 of 52) of the analyzed tumors, being found, for example, in 2 of the 12 tumors induced by SL3-3-Gln1 and 4 of the 13 tumors induced by SL3-3-Arg1,2 (Table 1). We therefore conclude that the introduced PBS mutations do not grossly affect tumorigenesis processes related to c-myc.

View larger version (112K): [in this window] [in a new window]   FIG. 2.   Southern blot of tumor DNAs digested with HindIII and probed for rearrangements in their TCR  loci (J2 probe). The arrow indicates the position of the nonrearranged germ line band.

To analyze if the introduced PBS mutations were genetically stable during in vivo replication and tumor induction, purified genomic DNAs from all tumors were subjected to PCR amplification with primers specific for the ecotropic MLV U3 region (primer 4, 5'-GATTCCCAGATGACCGGGGATC-3', which matches Akv positions 8179 to 8201) and gag (primer 2). The resulting ~750-bp amplicons were purified, and both strands of an ~250-bp region encompassing the PBS were directly sequenced with primers 1 and 3 (Table 2). While most of the sequence readouts were clean and allowed unambiguous assignments, direct sequencing of the PCR products from 14 of the tumors repeatedly resulted in poor-quality double sequences, consistent with sequencing of two different proviral templates. In these cases the PCR products were cloned into pGEM-T (Promega) and 8 to 12 individual subclones were sequenced. Surprisingly, SL3-3-Gln1 was the only virus found to replicate stably in all tumors analyzed whereas alterations within the PBS were detected in individual tumors induced by SL3-3-Pro, SL3-3-Lys3, and SL3-3-Arg1,2 (Table 2). A double T and C readout at position 8 for all of the analyzed Gln1 PBS sequences from tumor proviruses indicates that the SL3-3-Gln mutant may replicate with both tRNA₁^Gln and tRNA₂^Gln (Fig. 3), as was previously seen for Akv-MLV-derived vectors (24). wt SL3-3-Pro was ubiquitously found in tumors from mice originally injected with SL3-3-Pro, but in one tumor a PBS sequence matching tRNA^Gln was detected by subcloning the PCR product. The sequences flanking the Gln PBS contained a pattern of nucleotide substitutions, deletions, and insertions relative to the sequence of SL3-3-Pro virus originally injected and were highly homologous to previously identified proviral sequences endogenous to the murine genome (10, 29). A similar picture was seen with the lymphomas induced by the SL3-3-Arg1,2 mutant. SL3-3-Arg1,2 sequences were found again in all tumors, and one tumor contained additional proviruses harboring a glutamine PBS sequence (Table 2). Among the tumors taken from mice originally injected with SL3-3-Lys3, none contained only proviruses harboring the original PBS matching tRNA₃^Lys. In two of the tumors originally arising from infection with SL3-3-Lys3, the PBS had been altered to match the 3' 18 nucleotides of tRNA_1,2^Lys. The Lys3 PBS and Lys1,2 PBS differ at five positions only (Fig. 3). Since the sequences flanking the Lys1,2 PBS were SL3-3 specific, these altered viruses are likely to have originated from the binding of a tRNA_1,2^Lys directly to the Lys3 PBS sequence rather than from recombination with an endogenous virus harboring the Lys1,2 PBS. Interestingly, the tumor holding only a Lys1,2 PBS-containing SL3-3 mutant originated from the one SL3-3-Lys3-injected mouse in which disease induction was delayed to twice the average latency period. tRNA_1,2^Lys molecules are used as replication primers by visna (42) and spuma (28) viruses, and a PBS sequence matching tRNA_1,2^Lys has previously been identified in human endogenous retrovirus K (32). However, to our knowledge this represents the first report of an MLV replicating via tRNA_1,2^Lys. Since direct sequencing of PCR products amplified from tumors originating from mice injected with SL3-3-Lys3 resulted in mixed sequences in 12 of 13 tumors analyzed, PCR products from these tumors were cloned and individual subclones were sequenced. In 11 of 12 tumors, analysis of individual subclones revealed a mixture of proviral sequences carrying either the Lys3 or Gln PBS (Table 2). Whereas the sequences flanking the Lys3 PBS were identical to those in the original SL3-3-Lys3 mutant, all the sequences harboring a Gln PBS also contained the endogenous proviral sequence-characteristic pattern of substitutions, insertions, and deletions. In 1 of 12 of the tumors analyzed by sequencing of subclones, three PBS sequences, the Lys3, Gln, and Lys1,2 PBSs, were detected (Table 2). The exact nature of the recombinants is currently being investigated (26). To determine if second-site mutations were selectively induced as a function of replication with alternative tRNA primers, the sequences flanking the PBS (~250 bp in total) were inspected. Whereas single nucleotide substitutions were found in a number of proviruses, as were one 2-bp duplication and one 2-bp deletion, no evidence for a recurring tRNA-specific pattern of second-site mutations was found within the sequences flanking the PBS. Hence, we found no indications of molecular adaptation towards replication with alternative tRNA primers within the region analyzed. However, we cannot from these analyses dismiss the possibility of second-site mutations within the coding regions resulting from replication with tRNAs other than tRNA^Pro.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   (A) wt SL3-3 and mutant PBS-tRNA base pairings. Note that tRNA2Gln binding to the Gln1 PBS does not disrupt base pairing. (B) Potential base pairings between the Lys3 PBS and various tRNA species. Note that 14 base pairings may be formed between the Lys3 PBS and tRNA1,2Lys. Numbers to the right of the panels indicate numbers of bases paired.

Among the tumors originating from infection with SL3-3-Lys3, three were found to contain clonal rearrangements in the c-myc gene. Since recombinant proviruses containing Gln PBS sequences were detected in all three tumors, we wanted to analyze the PBS present in a c-myc-integrated provirus. With PCR primers specific for MLV gag and c-myc (43) an ~1.5-kb PCR fragment was generated and directly sequenced. The sequencing verified the presence of an SL3-3-Lys3 provirus integrated in the opposite transcriptional orientation relative to c-myc 1,415 bp upstream from the first c-myc exon (sequence information from GenBank accession no. M12345). SL3-3-Lys3 may therefore be involved in the tumorigenesis process in a manner similar to that of wt SL3-3, despite the observed delay in lymphoma induction (Fig. 1).

In conclusion, we find that the SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNA^Pro, although in vivo replication with alternative tRNA primers exhibits lowered replication kinetics. The reason for this finding remains unclear. While the results may reflect that MLV tRNA primer specificities act on the level of packaging, annealing, or RT-tRNA recognition, they may also reflect nonviral phenomena such as tRNA availability. In addition, alteration of the PBS sequence to match tRNA₃^Lys renders the mutant highly prone to recombination with endogenous MLV-like sequences. The results obtained in this study are in contrast to those of previous reports on PBS-modified retroviruses. While in this study none of the PBS mutants were found to revert to tRNA^Pro utilization, PBS-modified HIV-1 (11, 21, 45) and avian leukosis virus (47), analyzed by extended growth in cell culture, were found to eventually revert to the PBS sequence of the wt virus. Results from this study therefore sustain the notion of a less stringent usage of the MLV tRNA primer relative to those of other retroviruses and point to base pairings between the tRNA and the PBS sequence as the major determinant of replication primer selection. The PBS-modified viruses were found to induce malignant lymphomas indistinguishable from tumors induced by wt SL3-3 in terms of gross appearance, frequencies of genomic rearrangements within the TCR -chain and the c-myc locus, and an absence of genomic rearrangements within the Ig  light chain. Hence, we find no evidence that disruption of the negatively acting cis element originally identified in embryonal cells and overlapping the Pro PBS sequence significantly affected the viral tumorigenesis process in this model system. However, the complex pattern of molecular interactions underlying the development of neoplasms by MLV is still poorly understood, and it remains a formal possibility that negative effects of the Pro PBS sequence may reduce the appearance of revertants to Pro PBS. While several disease stages typical of MLV leukemogenesis, such as early viral replication within the bone marrow, rapid polyclonal expansion of preleukemic cells in the spleen, and proviral insertional mutagenesis and cis activation of cellular proto-oncogenes, may potentially be affected by the introduction of PBS mutations, the final effect of such PBS alterations cannot be predicted. We acknowledge, however, that the combination of highly pathogenic SL3-3 MLV isolates and a sensitive inbred mouse strain may have precluded the emergence of an altered pathogenicity pattern resulting from increased viral replication and transcriptional access in non-T cells.