Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

Immune status with empyema thoracis.

OBJECTIVE: To evaluate the humoral and cell mediated immune status of children with empyema thoracis. METHODS: Serum IgG, IgA, IgM, Complement C3 assay and cell mediated immunity (CMI) tests were performed in 33 patients of empyema thoracis, and 14 healthy age matched controls. RESULTS: The mean serum IgG and IgA levels in empyema thoracis and its subgroups were significantly raised as compared to controls. The overall values of IgG and IgA were 104% (p<0.001) and 114% (p<0.01) of normal mean, respectively. The mean serum IgM and complement C3 levels did not differ significantly in both the groups. The frequency of negative skin reaction to purified protein derivative (PPD) was significantly higher in children with empyema thoracis as compared to controls (p<0.05). The mean absolute lymphocyte count (ALC) was significantly decreased and serum adenosine deaminase (ADA) activity was significantly raised in empyema thoracis in comparison to controls. The overall ALC was 76.1% (p<0.01) and serum ADA activity was 169.4% (p<0.001) of normal mean, respectively. No significant differences were observed in the mean levels of immunoglobulins, complement C3 and CMI tests between pyothorax and pyopneumothorax and pleural fluid culture positive and negative cases. CONCLUSIONS: Thus, both humoral and cell mediated immunity were affected in empyema thoracis patients. However, CMI demonstrated more pronounced change in comparison to humoral immunity.

Dynamic copy choice: steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching.

We recently proposed a dynamic copy-choice model for retroviral recombination in which a steady state between the rates of polymerization and RNA degradation determines the frequency of reverse transcriptase (RT) template switching. The relative contributions of polymerase-dependent and polymerase-independent RNase H activities during reverse transcription and template switching in vivo have not been determined. We developed an in vivo trans-complementation assay in which direct repeat deletion through template switching reconstitutes a functional green fluorescent protein gene in a retroviral vector. Complementation in trans between murine leukemia virus Gag-Pol proteins lacking polymerase and RNase H activities restored viral replication. Because only polymerase-independent RNase H activity is present in this cell line, the relative roles of polymerase-dependent and -independent RNase H activities in template switching could be determined. We also analyzed double mutants possessing polymerase and RNase H mutations that increased and decreased template switching, respectively. The double mutants exhibited low template switching frequency, indicating that the RNase H mutations were dominant. Trans-complementation of the double mutants with polymerase-independent RNase H did not restore the high template switching frequency, indicating that polymerase-dependent RNase H activity was essential for the increased frequency of template switching. Additionally, trans-complementation of RNase H mutants in the presence and absence of hydroxyurea, which slows the rate of reverse transcription, showed that hydroxyurea increased template switching only when polymerase-dependent RNase H activity was present. This is, to our knowledge, the first demonstration of polymerase-dependent RNase H activity in vivo. These results provide strong evidence for a dynamic association between the rates of DNA polymerization and polymerase-dependent RNase H activity, which determines the frequency of in vivo template switching.

Design of retroviral vectors and helper cells for gene therapy.

During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered retroviruses are currently the most popular tool for gene delivery. Most of the systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles. Retroviridae is a family of retroviruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications. Currently, a variety of retroviruses that provide distinct advantages for gene delivery has been modified and used in clinical trials. In this review, the genome structures of oncoviruses, lentiviruses, and spumaviruses are reviewed and examples of vectors derived from these viruses are described. As with any delivery tool, the efficiency, the ability to target certain tissue or cell type, the expression of the gene of interest, and the safety of retroviral-based systems are important for successful application of gene therapy. Significant efforts have been dedicated to these areas of research in recent years. Various modifications have been made to retroviral-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety. The principles and design of these modifications are discussed in this review.

Role of murine leukemia virus reverse transcriptase deoxyribonucleoside triphosphate-binding site in retroviral replication and in vivo fidelity.

Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial beta-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1. 3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

Utilization of nonviral sequences for minus-strand DNA transfer and gene reconstitution during retroviral replication.

Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5' and 3' portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5' and 3' long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.

Effect of the murine leukemia virus extended packaging signal on the rates and locations of retroviral recombination.

Reverse transcriptase (RT) switches templates frequently during DNA synthesis; the acceptor template can be the same RNA (intramolecular) or the copackaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular template switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA was not accessible to the RT. To test our hypothesis, the murine leukemia virus (MLV) extended packaging signal (Psi(+)) containing a dimer linkage structure (DLS) was relocated from the 5' untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in this region. It was found that the overall maximum recombination rates of vectors with Psi(+) in the 5' UTR or Psi(+) between selectable markers were not drastically different. However, vectors with Psi(+) located between selectable markers reached a plateau of recombination rate at a shorter distance. This suggested a limited enhancement of recombination by Psi(+). The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5' Psi(+) including DLS did not undergo more recombination than expected from the size of the region. These experiments indicated that although the accessibility of the copackaged RNA was important in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addition, recombinants with multiple template switches were observed at a frequency much higher than expected, indicating the presence of high negative interference in the MLV-based system. This extends our observation with the spleen necrosis virus system and suggests that high negative interference may be a common phenomenon in retroviral recombination.

Structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching.

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.

Wild-type and YMDD mutant murine leukemia virus reverse transcriptases are resistant to 2',3'-dideoxy-3'-thiacytidine.

The antiretroviral nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.

Development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity.

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important for accuracy of DNA synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and the frequencies of inactivating mutations in lacZ were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of lacZ inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT (P < 0.005). Two mutations (V223I and Y598V) did not affect the frequency of lacZ inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.

Effect of distance between homologous sequences and 3' homology on the frequency of retroviral reverse transcriptase template switching.

Deletion of direct repeats in retroviral genomes provides an in vivo system for analysis of reverse transcriptase (RT) template switching. The effect of distance between direct repeats on the rate of deletion was determined for 16 murine leukemia virus (MLV)-based vectors containing a 701-bp direct repeat of overlapping fragments of the herpes simplex virus thymidine kinase gene (HTK). The direct repeats were separated by spacer fragments of various lengths (0.1 to 3.5 kb). Southern analysis of infected cells after one replication cycle indicated that all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing <1,500 bp between homologous sequences exhibited lower frequencies of deletion (37 to 82%). To analyze the pattern of locations at which RT switched templates, restriction site markers were introduced to divide the downstream direct repeat into five regions. RT switched templates within all five regions of the 701-bp direct repeat and the frequency of template switching was greater within the 5' regions in comparison to the 3' regions. The probability of RT switching templates within the 5' regions doubled when the MLV packaging sequence (Psi) was placed between the 701-bp direct repeats. However, Psi did not increase the rate of template switching for shorter direct repeats. These results indicate that linear distance between homologous sequences increases the rate of template switching and suggest that duplex formation between nascent DNA and homologous template sequences 3' of RT promote template switching.

Development of murine leukemia virus-based self-activating vectors that efficiently delete the selectable drug resistance gene during reverse transcription.

Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expression cassette during a single replication cycle at >99% efficiency.

Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate.

Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3'-azido-3'-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the lacZ mutant reporter gene were used. The frequencies of inactivating mutations introduced in the lacZ gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 microM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.

Relative rates of retroviral reverse transcriptase template switching during RNA- and DNA-dependent DNA synthesis.

Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site (att) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and att site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.

Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference.

Homologous recombination and deletions occur during retroviral replication when reverse transcriptase switches templates. While recombination occurs solely by intermolecular template switching (between copackaged RNAs), deletions can occur by an intermolecular or an intramolecular template switch (within the same RNA). To directly compare the rates of intramolecular and intermolecular template switching, two spleen necrosis virus-based vectors were constructed. Each vector contained a 110-bp direct repeat that was previously shown to delete at a high rate. The 110-bp direct repeat was flanked by two different sets of restriction site markers. These vectors were used to form heterozygotic virions containing RNAs of each parental vector, from which recombinant viruses were generated. By analyses of the markers flanking the direct repeats in recombinant and nonrecombinant proviruses, the rates of intramolecular and intermolecular template switching were determined. The results of these analyses indicate that intramolecular template switching is much more efficient than intermolecular template switching and that direct repeat deletions occur primarily through intramolecular template switching events. These studies also indicate that retroviral recombination occurs within a distinct viral subpopulation and exhibits high negative interference, whereby the selection of one recombination event increases the probability that a second recombination event will be observed.

Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

Utilization of nonhomologous minus-strand DNA transfer to generate recombinant retroviruses.

During reverse transcription, minus-strand DNA transfer connects the sequences located at the two ends of the viral RNA to generate a long terminal repeat. It is thought that the homology in the repeat (R) regions located at the two ends of the viral RNA sequences facilitate minus-strand DNA transfer. In this report, the effects of diminished R-region homology on DNA synthesis and virus titer were examined. A retrovirus vector, PY31, was constructed to contain the 5' and 3' cis-acting elements from Moloney murine sarcoma virus and spleen necrosis virus. These two viruses are genetically distinct, and the two R regions contain little homology. In one round of replication, the PY31 titer was approximately 3,000-fold lower than that of a control vector with highly homologous R regions. The molecular characteristics of the junctions of minus-strand DNA transfer were analyzed in both unintegrated DNA and integrated proviruses. Short stretches of homology were found at the transfer junctions and were likely to be used to facilitate minus-strand DNA transfer. Both minus-strand strong-stop DNA and weak-stop DNA were observed to mediate strand transfer. The ability of PY31 to complete reverse transcription indicates that minus-strand DNA transfer can be used to join sequences from two different viruses to form recombinant viruses. These results suggest the provocative possibility that genetically distinct viruses can interact through this mechanism.

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively.

Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNA.

A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA synthesis (internally initiated DNA). Strand transfer of internally initiated DNA would result in "dead" viral DNA that lacks the att site needed for integration. Strand transfer of the internally initiated DNA could occur if DNA synthesis failed to initiate at the PPT or if the PPT-primed DNA was displaced before strand transfer. We sought to determine the efficiency of DNA synthesis initiating at the PPT and the proportions of PPT-primed DNA and internally initiated DNAs that are utilized for strand transfer. We constructed spleen necrosis virus-based retroviral vectors containing an internal PPT and an att site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were determined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and that internally initiated DNAs are rarely utilized for strand transfer. We hypothesize that efficient synthesis and strand transfer of PPT-primed DNA evolved to prevent lethal strand transfers of internally initiated DNAs.