Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
Results
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
Conclusion
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

The role of jak2a in zebrafish hematopoiesis

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2617V>F) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2aUTR-MO) and splice-site junction (jak2aSS-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2aca). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1+ (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, he1 and ßhe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2aca significantly increased erythropoiesis and expression of gata1, he1 and ßhe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.

Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T_reg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4⁺ T_reg that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4⁺CD25⁺ T_reg, CD4⁺ T_reg induced by proinsulin DNA were both CD25⁺ and CD25^– and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T_reg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T_reg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.

Topotecan is a substrate for multidrug resistance associated protein 4

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both doselimiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated Km of 1.66 mM and Vmax of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 μM) for 24 hr, celecoxib (50 mM), or MK-571 (100 mM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POMPMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.

Human multidrug resistance associated protein 4 resitance to camtothecins

Purpose The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included.
Methods HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods.
Results Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine.
Conclusions Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.

One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond water metagenome

Metagenomics provides culture-independent access to gene pool of the whole microbial communities. To identify genes responsible for salt tolerance in unculturable bacteria, Escherichia coli clones were enriched with an ability to grow at inhibitory NaCl concentrations (750 mM) from a pond water metagenomic library. From two unique clones, genes encoding for proteins with similarity to a putative general stress protein (GspM) harbouring GsiB domain and a putative enoyl-CoA hydratase (EchM) were identified to be responsible for salt tolerance. The gspM was expressed by its native promoter whereas the echM was expressed from the lacZ promoter of the plasmid. EchM was overexpressed with a hexahistidyl tag. Purified EchM showed crotonyl-CoA hydratase activity. These genes have potential application in generating salt tolerant recombinant bacteria or transgenic plants.

Aspects of the production of viscous capsular material by the yoghurt starter, streptococcus thermophilus

The technology of modern fermented milk production is not complicated and relies largely on the characteristics of the microorganisms used in its manufacture. Biochemical substances excreted by the starter cultures contribute to the chemical, physical and organoleptic properties of cultured milks. Chemical and organoleptic properties of yoghurt starter cultures have been widely studied over several decades. Conversely the biosynthetic processes and genetic control of the production of viscous extracellular material (slime) by selected thermophillic streptococci is still insufficiently understood. This study attempted to elucidate physiological aspects and the genetic control of slime production. An attempt to chemically induce ropiness was also preformed. Twenty strains of Gram positive, thermo-tolerant, milk dotting, catalase negative cocci were collected from a variety of sources. All strains were identified as Streptococcus thermophilus. Four of the isolates were identified as capable of producing an extracellular, ‘ropy’ capsular material. A negative staining method for highlighting capsular material under light microscopy was described. Ropy isolates displayed thick capsular zones of between 6-8 μm. The isolates graded as non-ropy produced only small capsular zones (less than 2 μm); two variants displayed no capsular material. Instability of the ropy phenotype during subculture and prolonged storage was described for all four ropy isolates at varied temperatures. Instability during transfer was reported as moderate with a loss of no more than 45% of ropy colonies after 15 subcultures at 48°C A significant increase in instability, during transfer, associated with an increase in incubation temperature (37-48°C) was also reported. Prolonged storage of ropy variants over ten days resulted in a drop in the number of ropy colonies. The loss was minimal when cultures were stored at 8°C, but excessive (approaching 100%) at 37°C This suggested the presence of capsular degradative substances. Analysis of the plasmid profiles of 20 strains identified only two strains harboured plasmid DNA. All plasmids were small, less than 23kilobases, and each strain possessed a single plasmid species. Only one ropy strain contained plasmid DNA that was shown, with the aid of curing experiments, not to be linked to production of the ropy phenotype. The amino acid analogue p-fluoro-DL-phenylalanine was unsuccessful in generating ropy colonies from non-ropy variants of Streptococcus thermophilus at low concentrations. Some technological considerations for the use of ropy variants of Streptococcus thermophilus in yoghurt starter cultures were made.

Effects of survivin antagonists of growth of established tumors and B7-1 immunogene therapy

Background: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. Methods: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. Results: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P = .006 and P = .0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P = .004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. Conclusion: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.

Mouse B7-H3 induces antitumor immunity

Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10-9 M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit C AT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat . Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease ( p 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven C AT expression, while TNF- alpha -enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat /TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1.

Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.