scholarly journals RNase H Cleavage of the 5′ End of the Human Immunodeficiency Virus Type 1 Genome

2001 ◽  
Vol 75 (23) ◽  
pp. 11874-11880 ◽  
Author(s):  
Hong-Qiang Gao ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Strand Transfer ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The synthesis of retroviral DNA is initiated near the 5′ end of the RNA. DNA synthesis is transferred from the 5′ end to the 3′ end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5′ cap, to investigate the rules governing RNase H cleavage at the 5′ end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.

scholarly journals Mutations in the 5′ End of the Human Immunodeficiency Virus Type 1 Polypurine Tract Affect RNase H Cleavage Specificity and Virus Titer

2003 ◽  
Vol 77 (20) ◽  
pp. 11150-11157 ◽  
Author(s):  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stefan G. Sarafianos ◽  
W. Gregory Alvord ◽  
Edward Arnold ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Titer ◽  
Rnase H ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5′ end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5′ end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5′ end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by Degradation Products of Ceftazidime

1997 ◽  
Vol 8 (4) ◽  
pp. 353-362 ◽  
Author(s):  
SW Baertschi ◽  
AS Cantrell ◽  
MT Kuhfeld ◽  
U Lorenz ◽  
DB Boyd ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Degradation Products ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Hiv 1

Previous work by Hafkemeyer et al. (1991) [ Nucleic Acids Research19: 4059–4065] indicated that a degradation product of ceftazidime, termed HP 0.35, was active against the RNase H activity of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptase (RT) in vitro. Attempting to repeat these results, we isolated HP 0.35 from an aqueous degradation of ceftazidime and, after careful purification, we found HP 0.35 to be essentially inactive against both the polymerase and RNase H domains of HIV-1 RT (IC50 of >100 μg mL−1). During the investigation we discovered that polymeric degradation products of ceftazidime inhibited both the polymerase and, to a greater extent, the RNase H activities of HIV-1 RT in vitro (IC50 approximately 0.1 and 0.01 μg mL−1, respectively). Subjecting HP 0.35 to conditions under which it could polymerize induced inhibitory activity similar to that of the polymeric ceftazidime degradation products. It is proposed that the previously reported activity of HP 0.35 may have resulted from the presence of low levels of polymeric material either from incomplete purification or from polymerization of HP 0.35 during storage or in vitro testing.

scholarly journals Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

2000 ◽  
Vol 74 (20) ◽  
pp. 9668-9679 ◽  
Author(s):  
Christine Smith Snyder ◽  
Monica J. Roth
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Strand Transfer ◽  
Vitro Assay ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Truncated tRNA-DNA mimics were examined in an in vitro assay for second-strand transfer during human immunodeficiency virus type 1 (HIV-1) reverse transcription. Strand transfer in this system requires the progressive degradation of the RNA within the 18-mer tRNA-DNA (plus-strand strong stop DNA) intermediate to products approximately 8 nucleotides in length. The ability of the truncated substrates to substitute for directional processing by RNase H or reverse transcriptase (RT) was examined. Using wild-type HIV-1 RT, substrates which truncated the 5′ end of the tRNA primer by 6, 9, and 12 nucleotides (Δ6, Δ9, and Δ12, respectively) were recognized by RNase H and resulted in strand transfer. An overlap of 5 nucleotides between the acceptor and newly synthesized DNA template was sufficient for strand transfer. The mutant RT, E478Q correctly catalyzed the initial cleavage of the 18-mer tRNA-DNA mimic in the presence of Mn2+; however, no directional processing was observed. In contrast, no RNase H activity was observed with the Δ6, Δ9, and Δ12 substrates with E478Q RT in this strand transfer assay. However, when complemented with Escherichia coli RNase H, E478Q RT supported strand transfer with the truncated substrates. E478Q RT did cleave the truncated forms of the substrates, Δ6, Δ9, and Δ12, in a polymerase-independent assay. The size requirements of the substrates which were cleaved by the polymerase-independent RNase H activity of E478Q RT are defined.

scholarly journals Molecular Requirements for Human Immunodeficiency Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and Endogenous Reverse Transcription Systems

1999 ◽  
Vol 73 (6) ◽  
pp. 4794-4805 ◽  
Author(s):  
Tiyun Wu ◽  
Jianhui Guo ◽  
Julian Bess ◽  
Louis E. Henderson ◽  
Judith G. Levin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rnase H ◽  
Primer Binding Site ◽  
Strand Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3 Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3′ end of tRNA3 Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3′ rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3′-terminal bases by RNase H (added either in cis ortrans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes

2003 ◽  
Vol 77 (1) ◽  
pp. 291-300 ◽  
Author(s):  
L. Musey ◽  
Y. Ding ◽  
J. Cao ◽  
J. Lee ◽  
C. Galloway ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytotoxic T Lymphocyte ◽  
Infected Individual ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8+ cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8+ CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRβ VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.

scholarly journals Use of Coreceptors Other Than CCR5 by Non-Syncytium-Inducing Adult and Pediatric Isolates of Human Immunodeficiency Virus Type 1 Is Rare In Vitro

1998 ◽  
Vol 72 (11) ◽  
pp. 9337-9344 ◽  
Author(s):  
Yi-jun Zhang ◽  
Tatjana Dragic ◽  
Yunzhen Cao ◽  
Leondios Kostrikis ◽  
Douglas S. Kwon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Entry ◽  
Infected Infant ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have tested a panel of pediatric and adult human immunodeficiency virus type 1 (HIV-1) primary isolates for the ability to employ the following proteins as coreceptors during viral entry: CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CXCR4, Bonzo, BOB, GPR1, V28, US28, and APJ. Most non-syncytium-inducing isolates could utilize only CCR5. All syncytium-inducing viruses used CXCR4, some also employed V28, and one (DH123) used CCR8 and APJ as well. A longitudinal series of HIV-1 subtype B isolates from an infected infant and its mother utilized Bonzo efficiently, as well as CCR5. The maternal isolates, which were syncytium inducing, also used CXCR4, CCR8, V28, and APJ.

An Approach towards the Synthesis of Potential Metal-Chelating TSAO-T Derivatives as Bidentate Inhibitors of Human Immunodeficiency virus Type 1 Reverse Transcriptase

1998 ◽  
Vol 9 (5) ◽  
pp. 412-421 ◽  
Author(s):  
C Chamorro ◽  
M-J Camarasa ◽  
M-J Pérez-Pérez ◽  
E de Clercq ◽  
J Balzarini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Parent Compound ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

Novel derivatives of the potent human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor TSAO-T have been designed, synthesized and tested for their in vitro antiretro-viral activity against HIV. These TSAO-T derivatives have been designed as potential bidentate inhibitors of HIV-1 RT, which combine in their structure the functionality of a non-nucleoside RT inhibitor (TSAO-T) and a bivalent ion-chelating moiety (a β-diketone moiety) linked through an appropriate spacer to the N-3 of thymine of TSAO-T . Some of the new compounds have an anti-HIV-1 activity comparable to that of the parent compound TSAO-T, but display a markedly increased antiviral selectivity. There was a clear relationship between antiviral activity and the length of the spacer group that links the TSAO molecule with the chelating moiety. A shorter spacer invariably resulted in increased antiviral potency. None of the TSAO-T derivatives were endowed with anti-HIV-2 activity.

Elevated Levels of Serum-Soluble CD14 in Human Immunodeficiency Virus Type 1 (HIV-1) Infection: Correlation to Disease Progression and Clinical Events

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2084-2092 ◽  
Author(s):  
Egil Lien ◽  
Pål Aukrust ◽  
Anders Sundan ◽  
Fredrik Müller ◽  
Stig S. Frøland ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Disease Progression ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Number ◽  
Clinical Events ◽  
Immunodeficiency Virus ◽  
Hiv 1

Abstract Soluble (s) CD14, a marker for monocyte/macrophage activation and a mediator of bacterial lipopolysaccharide (LPS) action, was elevated in serum from human immunodeficiency virus type 1 (HIV- 1)-infected individuals (n = 92) compared with seronegative controls. The highest levels were found in patients with advanced clinical and immunological disease. Patients with ongoing clinical events had significantly higher sCD14 levels than symptomatic HIV-1-infected individuals without clinical events, with especially elevated levels in patients infected with Mycobacterium avium complex (MAC). On longitudinal testing of patients (n = 26) with less than 100 × 106CD4 lymphocytes/L at baseline, we found that increasing sCD14 serum concentrations per time unit were associated with death, whereas no differences in CD4 cell number decrease were found between survivors and nonsurvivors. In vitro studies showed that HIV-1 glycoprotein 120 and purified protein derivative (PPD) from M avium (MAC-PPD) stimulated normal monocytes to release sCD14. Furthermore, MAC-PPD induced tumor necrosis factor (TNF) release from monocytes through interactions with CD14 and, importantly, the addition of sCD14 enhanced this MAC-PPD stimulatory effect. Our findings suggest that the CD14 molecule may be involved in the immunopathogenesis of HIV-1 infection, and it is conceivable that serial determination of sCD14 may give useful predictive information concerning disease progression and survival in HIV-1-infected patients. © 1998 by The American Society of Hematology.

scholarly journals Separation of Human Immunodeficiency Virus Type 1 Replication from nef-Mediated Pathogenesis in the Human Thymus

2001 ◽  
Vol 75 (8) ◽  
pp. 3916-3924 ◽  
Author(s):  
Karen M. Duus ◽  
Eric D. Miller ◽  
Jonathan A. Smith ◽  
Grigoriy I. Kovalev ◽  
Lishan Su
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
In Vivo Model ◽  
Immunodeficiency Virus ◽  
Pathogenic Activity ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is frequently attenuated after long-term culture in vitro. The attenuation process probably involves mutations of functions required for replication and pathogenicity in vivo. Analysis of attenuated HIV-1 for replication and pathogenicity in vivo will help to define these functions. In this study, we examined the pathogenicity of an attenuated HIV-1 isolate in a laboratory worker accidentally exposed to a laboratory-adapted HIV-1 isolate. Using heterochimeric SCID-hu Thy/Liv mice as an in vivo model, we previously defined HIV-1 env determinants (HXB/LW) that reverted to replicate in vivo (L. Su, H. Kaneshima, M. L. Bonyhadi, R. Lee, J. Auten, A. Wolf, B. Du, L. Rabin, B. H. Hahn, E. Terwilliger, and J. M. McCune, Virology 227:46–52, 1997). Here we further demonstrate that HIV-1 replication in vivo can be separated from its pathogenic activity, in that the HXB/LW virus replicated to high levels in SCID-hu Thy/Liv mice, with no significant thymocyte depletion. Restoration of the nef gene in the recombinant HXB/LW genome restored its pathogenic activity, with no significant effect on HIV-1 replication in the thymus. Our results suggest that in vitro-attenuated HIV-1 lacks determinants for pathogenicity as well as for replication in vivo. Our data indicate that (i) the replication defect can be recovered in vivo by mutations in the envgene, without an associated pathogenic phenotype, and (ii)nef can function in the HXB/LW clone as a pathogenic factor that does not enhance HIV-1 replication in the thymus. Furthermore, the HXB/LW virus may be used to study mechanisms of HIV-1nef-mediated pathogenesis in vivo.

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