scholarly journals Treatment of Human Immunodeficiency Virus Type 1 Virions Depleted of Cyclophilin A by Natural Endogenous Reverse Transcription Restores Infectivity

2003 ◽  
Vol 77 (7) ◽  
pp. 4431-4434 ◽  
Author(s):  
Mahfuz Khan ◽  
Minerva Garcia-Barrio ◽  
Michael D. Powell
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Cyclosporine A ◽  
Reverse Transcription ◽  
Cyclophilin A ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Cypa Binding

ABSTRACT We have previously shown that virions with nef deleted can be restored to wild-type infectivity by treatment to induce natural endogenous reverse transcription (NERT). Since Nef and cyclophilin A (CyPA) appear to act in similar ways on postentry events, we determined whether NERT treatment would restore infectivity to virions depleted of CyPA. Our results show that the infectivity of virions depleted of CyPA by treatment with cyclosporine A could be restored by NERT treatment, while mutants in the CyPA binding loop of capsid could only be partially restored. These results suggest that CyPA is involved in some aspect of the uncoating process.

scholarly journals Escape and Compensation from Early HLA-B57-Mediated Cytotoxic T-Lymphocyte Pressure on Human Immunodeficiency Virus Type 1 Gag Alter Capsid Interactions with Cyclophilin A

2007 ◽  
Vol 81 (22) ◽  
pp. 12608-12618 ◽  
Author(s):  
Mark A. Brockman ◽  
Arne Schneidewind ◽  
Matthew Lahaie ◽  
Aaron Schmidt ◽  
Toshiyuki Miura ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Cyclophilin A ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8+ T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T242N substitution in the capsid protein is associated with upstream mutations at residues H219, I223, and M228 in the cyclophilin A (CypA)-binding loop in B57+ individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T242N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T242N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T242N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8+ T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.

scholarly journals A Mutation in Alpha Helix 3 of CA Renders Human Immunodeficiency Virus Type 1 Cyclosporin A Resistant and Dependent: Rescue by a Second-Site Substitution in a Distal Region of CA

2007 ◽  
Vol 81 (8) ◽  
pp. 3749-3756 ◽  
Author(s):  
Ruifeng Yang ◽  
Christopher Aiken
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Alpha Helix ◽  
Cell Protein ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT The replication of many isolates of human immunodeficiency virus type 1 (HIV-1) is enhanced by binding of the host cell protein cyclophilin A (CypA) to the viral capsid protein (CA). The immunosuppressive drug cyclosporine A (CsA) and its nonimmunosuppressive analogs bind with high affinity to CypA and inhibit HIV-1 replication. Previous studies have identified two mutations, A92E and G94D, in the CypA-binding loop of CA that confer the ability of HIV-1 to replicate in the presence of CsA. Interestingly, CsA stimulates the replication of HIV-1 mutants containing either the A92E or G94D substitution in some human cell lines. Here, we show that substitution of alanine for threonine at position 54 of CA (T54A) also confers HIV-1 resistance to and dependence on CsA. Like the previously identified CsA-resistant/dependent mutants, infection by the T54A mutant was stimulated by CsA in a target cell-specific manner. RNA interference-mediated reduction of CypA expression enhanced the permissiveness of HeLa cells to infection by the T54A mutant. A suppressor mutation, encoding a substitution of threonine for alanine at position 105 of CA (A105T), was identified through adaptation of the T54A mutant virus for growth in CEM cells. A105T rescued the impaired single-cycle infectivity and replication defects of both T54A and A92E mutants. These results indicate that CA determinants outside the CypA-binding loop can modulate the dependence of HIV-1 infection on CypA.

scholarly journals Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain

2005 ◽  
Vol 79 (3) ◽  
pp. 1470-1479 ◽  
Author(s):  
Isabel Scholz ◽  
Brian Arvidson ◽  
Doug Huseby ◽  
Eric Barklis
Keyword(s):  
Human Immunodeficiency Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Order Structures ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.

scholarly journals Replication of Phenotypically Mixed Human Immunodeficiency Virus Type 1 Virions Containing Catalytically Active and Catalytically Inactive Reverse Transcriptase

2001 ◽  
Vol 75 (14) ◽  
pp. 6537-6546 ◽  
Author(s):  
John G. Julias ◽  
Andrea L. Ferris ◽  
Paul L. Boyer ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Virus Replication ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Rnase H ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Catalytically Active

ABSTRACT The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.

scholarly journals Roles of Pr55gag and NCp7 in tRNA3Lys Genomic Placement and the Initiation Step of Reverse Transcription in Human Immunodeficiency Virus Type 1

2000 ◽  
Vol 74 (22) ◽  
pp. 10796-10800 ◽  
Author(s):  
Shan Cen ◽  
Ahmad Khorchid ◽  
Juliana Gabor ◽  
Liwei Rong ◽  
Mark A. Wainberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Initiation Step

ABSTRACT To study in vivo tRNA3 Lys genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR−) virus was used as the source of primer tRNA3 Lys/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA3 Lys, dCTP, were lower with PR− than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR− dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55 gag had no effect on initiation. These results indicate that while Pr55 gag is sufficient for tRNA3 Lys placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA3 Lys placement and initiation of reverse transcription.

scholarly journals Defective Replication in Human Immunodeficiency Virus Type 1 When Non-\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}Primers Are Used for Reverse Transcription

2005 ◽  
Vol 79 (14) ◽  
pp. 9081-9087 ◽  
Author(s):  
Min Wei ◽  
Shan Cen ◽  
Meijuan Niu ◽  
Fei Guo ◽  
Lawrence Kleiman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Pcr Analysis ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , the primer for reverse transcriptase in human immunodeficiency virus type 1 (HIV-1), anneals to the primer binding site (PBS) in HIV-1 RNA. It has been shown that altering the PBS and U5 regions upstream of the PBS in HIV-1 so as to be complementary to sequences in tRNAMet or tRNAHis will allow these tRNA species to be stably used as primers for reverse transcription. We have examined the replication of these mutant viruses in Sup-T1 cells. When Sup-T1 cells are infected by cocultivation with HIV-1-transfected 293T cells, viruses using tRNAHis or tRNAMet are produced at rates that are approximately 1/10 or 1/100, respectively, of rates for wild-type virions that use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} . When Sup-T1 cells are directly infected with equal amounts of these different viruses isolated from the culture supernatant of transfected 293T cells, virions using tRNAMet are produced at 1/100 the rate of wild-type viruses, and production of virions using tRNAHis is not detected. Both wild-type and mutant virions selectively package tRNALys only, and examination of the ability of total viral RNA to prime reverse transcription in vitro indicates a >80% reduction in the annealing of tRNAHis or tRNAMet to the mutant viral RNAs. PCR analysis of which of the three primer tRNAs is used indicates that only \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} is detected as primer in wild-type virions and only tRNAHis is detected as primer in virions containing a PBS complementary to tRNAHis, while the mutant viruses containing a PBS complementary to tRNAMet use both tRNAMet and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{1,2}^{Lys}\) \end{document} as primer tRNAs.

scholarly journals Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase

2003 ◽  
Vol 77 (23) ◽  
pp. 12592-12602 ◽  
Author(s):  
Shixing Tang ◽  
Tsutomu Murakami ◽  
Naiqian Cheng ◽  
Alasdair C. Steven ◽  
Eric O. Freed ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Capsid Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Cyclophilin A ◽  
Virus Infectivity ◽  
Immunodeficiency Virus

ABSTRACT We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

scholarly journals Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 81 (18) ◽  
pp. 10047-10054 ◽  
Author(s):  
Lori V. Coren ◽  
James A. Thomas ◽  
Elena Chertova ◽  
Raymond C. Sowder ◽  
Tracy D. Gagliardi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Mutational Analysis ◽  
Cleavage Sites ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6Gag proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9NC/SP2 protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8SP2/p6 or p15NC/SP2/p6 protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6Gag site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6Gag and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9NC/SP2 species in some aspect of integration.

scholarly journals The Role of Nucleocapsid and U5 Stem/A-Rich Loop Sequences in tRNA3Lys Genomic Placement and Initiation of Reverse Transcription in Human Immunodeficiency Virus Type 1

1998 ◽  
Vol 72 (5) ◽  
pp. 3907-3915 ◽  
Author(s):  
Yue Huang ◽  
Ahmad Khorchid ◽  
Juliana Gabor ◽  
Jing Wang ◽  
Xuguang Li ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Extracellular Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA3 Lys genomic placement, i.e., the in vivo placement of primer tRNA3 Lys on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA3 Lys to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA3 Lys occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55 gag and/or Pr160 gag-pol . One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160 gag-pol and tRNA3 Lys and prevented the genomic placement of tRNA3 Lys. Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160 gag-pol . For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160 gag-pol and tRNA3 Lyswas not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55 gag or Pr160 gag-pol . After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA3 Lys. This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A’s in the loop) did not affect the in vivo genomic placement of tRNA3 Lys but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA3 Lys, with a corresponding decrease in the presence of unextended and 2-base-extended tRNA3 Lys.

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