Retroviral Replication Cycle Research Articles

Retroviral recombination rates do not increase linearly with marker distance and are limited by the size of the recombining subpopulation.

Recombination occurs at high frequencies in all examined retroviruses. The previously determined homologous recombination rate in one retroviral replication cycle is 4% for markers 1.0 kb apart in spleen necrosis virus (SNV). This has often been used to suggest that approximately 30 to 40% of the replication-competent viruses with 7- to 10-kb genomes undergo recombination. These estimates were based on the untested assumption that a linear relationship exists between recombination rates and marker distances. To delineate this relationship, we constructed three sets of murine leukemia virus (MLV)-based vectors containing the neomycin phosphotransferase gene (neo) and the hygromycin phosphotransferase B gene (hygro). Each set contained one vector with a functional neo and an inactivated hygro and one vector with a functional hygro and an inactivated neo. The two inactivating mutations in the three sets of vectors were separated by 1.0, 1.9, and 7.1 kb. Recombination rates after one round of replication were 4.7, 7.4, and 8.2% with markers 1.0, 1.9, and 7.1 kb apart, respectively. Thus, the rate of homologous recombination with 1.0 kb of marker distance is similar in MLV and SNV. The recombination rate increases when the marker distance increases from 1.0 to 1.9 kb; however, the recombination rates with marker distances of 1.9 and 7.1 kb are not significantly different. These data refute the previous assumption that recombination is proportional to marker distance and define the maximum recombining population in retroviruses.

Integration of Visna Virus DNA Occurs and May Be Necessary for Productive Infection

Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host–virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host–U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells.

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.

Design of retroviral expression vectors.

Several characteristics of amphotropic murine retroviruses have made them useful as vectors for gene transfer with some distinct advantages over other methods of transduction First, the normal replication cycle of retroviruses includes integration of the viral genome into the host's chromosomal DNA, and these viruses have evolved a very efficient mechanism of stable gene transfer for this purpose Second, because the integration machinery uses the termini of the viral DNA as substrate, insertion of viral DNA takes place m a predictable manner with the long terminal repeat (LTR) sequences flanking the genes they carry. Third, retroviruses have a broad host range, which allows gene transfer and expression of foreign genes in many cell types, even including some cell types that are refractory to gene transfer by other means. Fourth, a cytopathic effect on infected cells is lacking, which is especially important for gene expression studies and the generation of stable cell lines Fifth, all retroviral proteins required for the assembly of infectious virions can be supplied in trans, thereby enabling the expression of exogenous genes up to approx 8 kbp. Finally, retroviral vectors can be conveniently manipulated in plasmid form and, as discussed below, are somewhat flexible in terms of vector design. There are, however, some limitations to the use of retroviral vectors. One limitation is that gene transfer only occurs when cells are actively dividing (1) A second limitation is that expression levels are only moderate compared to those achieved by other methods of gene transfer, owing m part to the low copy numbers of the integrated provirus generally achievable by these vectors. The first component of the retrovirus gene transfer system is the retroviral packaging cell line. Packaging cells provide all the retroviral proteins required for the encapsidation of the retroviral vector (in RNA form) into infectious

Structure and Function of the Human Immunodeficiency Virus Leader RNA

Publisher Summary This chapter deals with the structure and function of the leader transcript of human immunodeficiency virus (HIV-1) and HIV-2. Most of the RNA signals encoded by the untranslated leader RNA have specific nucleotide sequences critical for recognition and function, but there is accumulating evidence that their structural context can also be important. There is an intense effort to analyze the secondary structure of retroviral leader RNAs using a variety of methods, including biochemical analysis, free-energy minimization, sequence comparison, and mutant analysis. The chapter focuses primarily on the relationships between the structure of specific leader RNA motifs and their function in the retroviral life cycle. Among retroviruses, the lentiviruses have the most complex genome structure and expression strategy. The primate lentiviruses include the human and simian immunodeficiency viruses (SIV). Understanding the three-dimensional structure formed by the HIV leader RNA molecule, both free and in the ribonucleoprotein complex of virion, is crucial for analyzing its specific recognition by proteins and its interaction by other RNA molecules during virus replication. The number of HIV–SIV sequences now known is sufficiently large; hence, comparative analysis can be used effectively to deduce some of the basic design principles underlying HIV RNA structure. By understanding the molecular basis of the interactions that govern critical steps in the retroviral replication cycle, it may be possible to develop methods to intervene therapeutically in the process.

Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications.

Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp.

High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication.

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.

One retroviral RNA is sufficient for synthesis of viral DNA

We used previously characterized spleen necrosis virus-based retroviral vectors and helper cells to study the strand transfers that occur during the reverse-transcription phase of a single cycle of retroviral replication. The conditions used selected only for formation of an active provirus rather than for expression of multiple drug resistance markers. In nonrecombinant proviruses the minus- and plus-strand DNA primer transfers were almost completely intramolecular. However, as previously reported, recombinant proviruses contained approximately equal proportions of inter- and intramolecular minus-strand DNA primer transfers. Thus, we conclude that in the absence of recombination, one molecule of retroviral RNA is sufficient for viral DNA synthesis. Large deletions and deletions with insertions were detected primarily at a limited number of positions which appear to be hot spots for such events, the primer binding site and regions containing multiple inverted repeats. At these hot spots, the rate of deletions and deletions with insertions visible with PCR was about 10% per genome per replication cycle. Other deletions and deletions with insertions (detectable with PCR) occurred at a rate of about 0.5%/kb per replication cycle. Crossovers occurred at a rate of about 6%/kb per replication cycle under single-selection conditions. This rate is comparable to the rate that we reported previously under double-selection conditions, indicating that retroviral homologous recombination is not highly error prone. The combined rates of deletions and deletions with insertions at hot spots (10% per genome per replication cycle) and other sites (0.5%/kb per replication cycle) and the rate of crossovers (6%/kb per replication cycle) indicate that on average, full-size (10-kb) type C retroviruses undergo an additional or aberrant strand transfer about once per cycle of infection.

High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector

A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.

Rate and Mechanism of Nonhomologous Recombination During a Single Cycle of Retroviral Replication

Oncogenes discovered in retroviruses such as Rous sarcoma virus were generated by transduction of cellular proto-oncogenes into the viral genome. Several different kinds of junctions between the viral and proto-oncogene sequences have been found in different viruses. A system of retrovirus vectors and a protocol that mimicked this transduction during a single cycle of retrovirus replication was developed. The transduction involved the formation of a chimeric viral-cellular RNA, strand switching of the reverse transcription growing point from an infectious retrovirus to the chimeric RNA, and often a subsequent deletion during the rest of viral DNA synthesis. A short region of sequence identity was frequently used for the strand switching. The rate of this process was about 0.1 to 1 percent of the rate of homologous retroviral recombination.

Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase.

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.

GENERATION OF DIVERSITY IN RETROVIRUSES

Retroviruses are unique in that their propagation includes transfer of genetic information from RNA to DNA. Two enzymes (RT and RNA polymerase II) that participate in their replication process do not encode editing functions and are thus "error prone." Current estimates indicate that up to one nucleotide substitution per genome occurs per retrovirus replication cycle. In addition, rearrangements can occur during reverse transcription. These mutations result in viral populations in which the wild-type sequence can only be defined by consensus. Retroviruses are also unusual among viruses in their high recombination frequency, which is the result of the copackaging, and then reverse transcribing of two different RNA genomes in the same particle. Recombinants are formed during copying of RNA templates into DNA. With the ability to mutate and recombine genetic information at a higher rate, retroviral populations are poised to respond to selective forces, which may increase or decrease replication of particular genotypes. Interspecies transmission of retroviruses or retroviral genes also likely plays a role in generating diversity. Endogenous proviruses exist in the germline of many vertebrates; pedigrees indicate that recent infections of the germline have also occurred. Thus, the selective forces that mold the retroviral genome may be opposing: selection for efficient replication as exogenous viruses versus selection for passive replication as endogenous proviruses. The latter may be more advantageous over the course of evolution since the survival of the retrovirus is ensured by survival of the host organism. Segments of endogenous viruses may reappear in exogenous viruses through recombination and thus these endogenous sequences are perpetuated.

Studies of the mechanisms of action of the antiretroviral agents hypericin and pseudohypericin.

Administration of the aromatic polycyclic dione compounds hypericin or pseudohypericin to experimental animals provides protection from disease induced by retroviruses that give rise to acute, as well as slowly progressive, diseases. For example, survival from Friend virus-induced leukemia is significantly prolonged by both compounds, with hypericin showing the greater potency. Viremia induced by LP-BM5 murine immunodeficiency virus is markedly suppressed after infrequent dosage of either substance. These compounds affect the retroviral infection and replication cycle at least at two different points: (i) Assembly or processing of intact virions from infected cells was shown to be affected by hypericin. Electron microscopy of hypericin-treated, virus-producing cells revealed the production of particles containing immature or abnormally assembled cores, suggesting the compounds may interfere with processing of gag-encoded precursor polyproteins. The released virions contain no detectable activity of reverse transcriptase. (ii) Hypericin and pseudohypericin also directly inactivate mature and properly assembled retroviruses as determined by assays for reverse transcriptase and infectivity. Accumulating data from our laboratories suggest that these compounds inhibit retroviruses by unconventional mechanisms and that the potential therapeutic value of hypericin and pseudohypericin should be explored in diseases such as AIDS.

The 3' long terminal repeat of a transcribed yet defective endogenous retroviral sequence is a competent promoter of transcription.

Although actively transcribed and present as multiple genomic copies, a distinct class of endogenous murine leukemia virus-related sequence does not give rise to infectious virus. Since the long terminal repeat at the 3' terminus provides the transcriptional start site after reintegration, we determined the structure and potential promoter activity of that sequence obtained from cDNA of endogenous retroviral transcripts. These studies demonstrate that the distinctive 3' long terminal repeat sequence of these transcripts could serve as an effective promoter of transcription and, therefore, may not be the primary defect in the infectious cycle of retroviral replication but may result in the propagation of these endogenous retroviral sequences in the genome as retrotransposons.