Proviral DNA Synthesis Research Articles

Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Penetration into Target Cells by Synthetic Peptides Mimicking the N-Terminus of the HIV-1 Transmembrane Glycoprotein

To investigate the mechanism of action of the 22-amino-acid HIV fusion peptide on HIV infection, we studied its influence on virus adsorption and HIV-induced syncytium formation. The effect of the peptide preparations on the synthesis of viral antigens in HIV-infected cell cultures was determined by antigen capture assay, and the inhibition of proviral DNA synthesis was detected by hybridization with a HIV-specific oligonucleotide probe after PCR amplification. Fusion peptides inhibited HIV-induced syncytium formation and antigen production in lyric infected cells, and this effect was increased in conjugation with bovine serum albumin or with synthetic net-charged polymer by its C-terminus. The association of peptide with carrier by N-terminus, or with positive-charged polymer or gelatin completely abolished its effect on HIV infection. No peptide preparations influenced HIV-1 chronically infected cells. Because peptide preparations blocked the HIV-specific DNA synthesis 2 hr after infection without influencing virus adsorption and reverse transcription, we concluded that the block of infection occurred during the penetration of virions through the cell membrane. On the basis of results obtained we propose that our peptide preparations could be used for anti-HIV chemotherapy. The possibility of the existence of receptors for gp41 N-terminal region on target cell membrane is discussed.

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

HIV-1 proteinase is required for synthesis of pro-viral DNA

HIV-1 proteinase activity is thought to occur primarily post-integration by cleaving the viral Gag and Gag-Pol polyproteins. Its role in the pre-integration stages of viral replication, however, has not been studied in detail. Here we report that a synthetic peptide analogue, UK-88,947, which is a specific inhibitor of purified HIV-1 proteinase, inhibits the processing of the viral polyproteins in cultures of HIV-1 infected cells and prevents the formation of mature, infectious virions. Analysis of DNA from HIV-1 infected cells treated with UK-88,947 showed that viral DNA synthesis was inhibited when the compound was added to cultures one hour before infection. Similar results were obtained when AZT was used. Neither HIV-1 reverse transcriptase or the replication of FIV are inhibited by UK-88,947.

Retroviral Vectors Related to the Myeloproliferative Sarcoma Virus Allow Efficient Expression in Hematopoietic Stem and Precursor Cell Lines, but Retroviral Infection Is Reduced in More Primitive Cells

Retroviral vectors are considered to be the most suited vehicles for somatic gene therapy with hematopoietic stem cells as targets. Retrovirus-mediated gene transfer into differentiation-restricted hematopoietic precursor (FDC-P1, FDC-P2) and multipotent progenitor (stem) cell lines (FDC-Pmix) is inefficient. Two cellular restrictions are involved. One is specific for stem but not precursor cells and is at the level of transcription. Due to a unique property of the transcriptional control region of the myeloproliferative sarcoma virus (MPSV), vectors derived from MPSV are not affected by this block. The second restriction occurs before proviral DNA synthesis and integration. This inhibition of effective viral infection depends on the state of differentiation, being more pronounced in multipotent clonogenic blast cells. This block to retroviral infection affects all retroviral vectors tested.

Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene

A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication.

Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro

Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.

Two interferon sensitive steps in the replication cycle of rous sarcoma virus

De novo infection of Rous sarcoma virus (RSV) strains of receptor subgroups A, B, C, and D is inhibited by low doses of chick interferon. Adsorption and penetration into the cell are not or only marginally impaired by interferon treatment. Since the level of proviral DNA synthesis is strongly reduced in the interferon-treated cell it is concluded that uncoating or reverse transcription of the viral genome is inhibited. This inhibition of proviral DNA synthesis is not caused by an arrest of CEF in the stationary phase. Chronic infection of SR-RSV-A, -B, -C, but not SR-RSV-D is also sensitive to interferon. Chick interferon treatment (50 u/ml) also had no inhibitory effect on the amount of transcripts of the RSV-specific oncogene src or the cellular oncogenes src and myc.

Inhibition of HIV-1 proviral DNA synthesis and RNA accumulation by mismatched dsRNA

The antiviral activity of mismatched dsRNA of the form poly(I):poly(C 12-U) n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIs cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.

Broad spectrum antiretroviral activity of 2',3'-dideoxynucleosides.

Certain dideoxynucleosides have been shown to markedly inhibit the infectivity of human T-lymphotropic virus type III/lymphadenopathy-associated virus, the causative agent of acquired immunodeficiency syndrome (AIDS). Our present studies demonstrate that these drugs are broad spectrum antiretroviral agents capable of inhibiting the infectivity of evolutionarily divergent mammalian type C and animal lentiviruses. Under some conditions, virus infectivity could be inhibited by more than six orders of magnitude. However, the potency of these agents was shown to be greatly influenced by cell-specified determinants. Drug exposure during the initial 24 hr was almost as effective as prolonged treatment on the inhibition of a single cycle of virus infection and expression. Moreover, virus infection was shown directly to be inhibited at the level of proviral DNA synthesis. Thus the time period during which reverse transcription and provirus integration occur is the critical period required for drug action. Our findings have implications concerning strategies to be considered in attempts to utilize 2',3'-dideoxynucleosides in control and treatment of retrovirus-induced diseases of animals and humans.

Generation of infectious Moloney murine leukemia viruses with deletions in the U3 portion of the long terminal repeat.

Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20- to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.

Generation of infectious Moloney murine leukemia viruses with deletions in the U3 portion of the long terminal repeat.

Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20- to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.

Synthesis of proviral DNA in inbred mouse-derived clones of cells expressing different Fv-1 phenotypes.

Formation of proviral DNAs by B-tropic murine leukaemia viruses (MLVs) was examined in N-type and dually permissive mutant cells derived from two inbred mouse strains, DDD and G, both of which are N-type. In the N-type cells, formation of circular proviral DNA was strongly suppressed relative to that of linear DNA. Mutation resulting in loss of the N-type Fv-1 restriction resulted in efficient formation of circular DNA by the previously restricted B-tropic MLV. This showed that Fv-1 restriction and inhibition of closed circular DNA formation were controlled by the same gene. The efficiency of formation of circular proviral DNA by the defective Kirsten murine sarcoma virus was determined by the tropism of the helper virus.

Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

Effect of interferon on the exogenous friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65 gag , and glycoprotein precursor, gPr85 env , to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65 gag , was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.

Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

The murine gene Fv-1 predominantly controls the outcome of infection by murine ecotropic retroviruses. The inhibition of virus replication by the Fv-1 gene product has been determined to be at an early stage in virus replication. Mechanistically, its effect appears to be on the accumulation of unintegrated proviral DNA or its integration or both. We investigated the synthesis of unintegrated proviral DNA, using several clones of B-, N-, or NB-tropic Friend murine leukemia virus. Our results indicate that the accumulation of B-tropic proviral DNA in NIH cells may be inhibited at either the level of linear (form III) or covalently closed circular DNA (form I), depending upon the degree of restriction of the clone of virus used. We confirmed that there is an effect of the Fv-1 gene on the accumulation of form I DNA of either B- or N-tropic Friend murine leukemia virus. However, the decrease in infectious centers effected by the Fv-1 gene did not correlate quantitatively with the effect on form I proviral DNA produced by N-tropic Friend murine leukemia virus in nonpermissive cells. Lastly, we demonstrated in nonpermissively infected NIH cells that a rapidly migrating doublet of viral DNA is formed.

Studies on the restriction of ecotropic murine retrovirus replication in mouse teratocarcinoma cells.

Retrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.

Analysis of avian myeloblastosis viral RNA and in vitro synthesis of proviral DNA.

Two virus-specific RNA species of 7.5 and 7.0 kilobases have been identified in avian myeloblastosis virus (AMV) by denaturing gel electrophoresis and blot hybridization analysis, and they were found to be in a 10:1 ratio. The individual RNAs direccted the cell-free synthesis of the 76,000-dalton "gag" protein and the 180,000-dalton "gal-pol" protein, thereby demonstrating 5' sequence homology of approximately 4.9 kilobases between the two species. Synthesis of these two precursor proteins by the AMV genome indicates structural differences between AMV and other avian acute leukemia viruses. The two viral RNAs were transcribed into complete cDNA copies with AMV DNA polymerase. Linear proviruses were found to be 90--100% resistant to S1 nuclease. Analysis of single-stranded transcripts demonstrated two distinct species of 2.6 and 2.3 x 10(6) daltons, and analysis of duplexes formed from the single-stranded transcripts demonstrated species of 5.2 and 4.0 x 10(6) daltons.

Fusion of normal primate cells: A common biological property of the D-type retroviruses

Three different D-type oncoviruses, Mason-Pfizer monkey virus (M-PMV), squirrel monkey retrovirus (SMRV), and langur virus (PO-1-Lu), have been shown to induce cell fusion in different nontransformed human and nonhuman primate cell lines. This therefore represents the first report that cell fusion is a common property of all three D-type retroviruses. In contrast, primate and feline C-type retroviruses (baboon endogenous virus, woolly monkey leukemia virus, and RD-114) fail to induce syncytia in parallel experiments. Inhibitor studies indicate that the three D-type oncoviruses induce multinucleate cell formation by a common mechanism, different from that of either the paramyxoviruses or the syncytia-forming foamy viruses. Increasing doses of ultraviolet irradiation reduce the syncytia-forming ability of these viruses with single-hit kinetics. However, proviral DNA synthesis is not required for cell fusion since the addition of cytosine arabinoside has no significant effect on multinucleate cell formation at concentrations which inhibit virus replication. This result indicates that virus replication is not required for multinucleate cell formation and that the two are independent events of virus infection. The addition of cycloheximide during the first 12 hr of infection, on the other hand, significantly reduces cell fusion, indicating that de novo protein synthesis is required. The possibility that cell fusion involves translation of input viral RNA is discussed. Pre-infection of target cells with the replicating component of M-PMV blocks fusion by all three D-type viruses. The degree of interference observed correlates with the molecular relatedness of the three viruses.