scholarly journals Rescue of Multiple Viral Functions by a Second-Site Suppressor of a Human Immunodeficiency Virus Type 1 Nucleocapsid Mutation

2000 ◽  
Vol 74 (9) ◽  
pp. 4273-4283 ◽  
Author(s):  
Andrea Cimarelli ◽  
Sara Sandin ◽  
Stefan Höglund ◽  
Jeremy Luban
Keyword(s):  
Serial Passage ◽  
Full Length ◽  
Compensatory Mutation ◽  
Rna Packaging ◽  
Viral Dna ◽  
Stem Loop ◽  
The Face ◽  
A Minor ◽  
Hiv 1

ABSTRACT Human immunodeficiency type 1 (HIV-1) bearing the nucleocapsid (NC) mutation R10A/K11A is replication defective. After serial passage of the mutant virus in tissue culture, we isolated a revertant that retained the original mutation. It had acquired, in addition, a new mutation (E21K) that was formally demonstrated to be sufficient for restoration of viral replication. Detailed analysis of the replication defect of R10A/K11A revealed a threefold reduction in virion yield and a fivefold reduction in packaging of viral genomic RNA. Real-time PCR was then used to quantitate viral DNA synthesis following infection of Jurkat T cells. After adjustment for the assembly and packaging defects, a minor (twofold) reduction in synthesis of either strong-stop, full-length linear DNA or 2-LTR circles was observed with R10A/K11A virions, indicating that reverse transcription and nuclear transport of the viral genome were largely intact. However, after adjustment for the amounts of full-length or 2-LTR circles produced, R10A/K11A virions were at least 10-fold less infectious than wild type, indicating that viral DNA produced by the R10A/K11A mutant failed to integrate. Each of the above-mentioned defects was corrected by introduction of the second-site compensatory mutation E21K. These results demonstrate that the replication defect of mutant R10A/K11A can be explained by impairment at multiple steps in the viral life cycle, most important among them being integration and RNA packaging. The E21K mutation is predicted to restore positive charge to the face of the R10A/K11A mutant NC protein that interacts with the HIV-1 SL3 RNA stem-loop, emphasizing the importance of NC basic residues for HIV-1 replication.

scholarly journals Possible Role of Dimerization in Human Immunodeficiency Virus Type 1 Genome RNA Packaging

2003 ◽  
Vol 77 (7) ◽  
pp. 4060-4069 ◽  
Author(s):  
Jun-Ichi Sakuragi ◽  
Shigeharu Ueda ◽  
Aikichi Iwamoto ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Packaging ◽  
Dimer Formation ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Lower Stem ◽  
Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5′ region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.

scholarly journals The Human Immunodeficiency Virus Type 1 TAR RNA Upper Stem-Loop Plays Distinct Roles in Reverse Transcription and RNA Packaging

2000 ◽  
Vol 74 (12) ◽  
pp. 5639-5646 ◽  
Author(s):  
David Harrich ◽  
C. William Hooker ◽  
Emma Parry
Keyword(s):  
Reverse Transcription ◽  
Transcription Initiation ◽  
Loop Structure ◽  
Rna Packaging ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

scholarly journals Human Immunodeficiency Virus Type 1 Nucleocapsid Zn2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA

2003 ◽  
Vol 77 (2) ◽  
pp. 1469-1480 ◽  
Author(s):  
James S. Buckman ◽  
William J. Bosche ◽  
Robert J. Gorelick
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Full Length ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn2+ finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn2+ fingers (NCH23C and NCH44C), an integrase mutant (IND116N), a double mutant (NCH23C/IND116N), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn2+ finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.

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.

Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design

2004 ◽  
Vol 18 (12) ◽  
pp. 739-760 ◽  
Author(s):  
Rajeshri G. Karki ◽  
Yun Tang ◽  
Terrence R. Burke ◽  
Marc C. Nicklaus
Keyword(s):  
Drug Design ◽  
Full Length ◽  
Viral Dna ◽  
Anti Hiv ◽  
Hiv 1

scholarly journals Mutations in the Human Immunodeficiency Virus Type 1 Integrase D,D(35)E Motif Do Not Eliminate Provirus Formation

1998 ◽  
Vol 72 (6) ◽  
pp. 4678-4685 ◽  
Author(s):  
Meenakshi Gaur ◽  
Andrew D. Leavitt
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Genome ◽  
Viral Dna ◽  
Infectious Titer ◽  
Immunodeficiency Virus ◽  
Acidic Residues ◽  
Hiv 1

ABSTRACT The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3′-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 103- to 104-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721–728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per μg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3′-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.

Virtual screening application of a model of full-length HIV-1 integrase complexed with viral DNA

2007 ◽  
Vol 17 (19) ◽  
pp. 5361-5365 ◽  
Author(s):  
Chenzhong Liao ◽  
Rajeshri G. Karki ◽  
Christophe Marchand ◽  
Yves Pommier ◽  
Marc C. Nicklaus
Keyword(s):  
Virtual Screening ◽  
Full Length ◽  
Viral Dna ◽  
Hiv 1

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

2007 ◽  
Vol 81 (22) ◽  
pp. 12189-12199 ◽  
Author(s):  
Krishan K. Pandey ◽  
Sibes Bera ◽  
Jacob Zahm ◽  
Ajaykumar Vora ◽  
Kara Stillmock ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Dependent Manner ◽  
Strand Transfer ◽  
Viral Dna ◽  
Target Dna ◽  
Immunodeficiency Virus ◽  
Dna Ends ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

scholarly journals Dissociation of Genome Dimerization from Packaging Functions and Virion Maturation of Human Immunodeficiency Virus Type 1

2002 ◽  
Vol 76 (3) ◽  
pp. 959-967 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Aikichi Iwamoto ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Rna ◽  
Rna Packaging ◽  
Virus Particles ◽  
Immunodeficiency Virus ◽  
Rna Interaction ◽  
Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is thought to play important roles at various stages of the virus life cycle. Recently we showed that the DIS/DLS region affects RNA-RNA interaction in intact virus particles, by demonstrating that duplication of the region in viral RNA caused the production of virus particles containing partially monomeric RNAs. We have extended this finding and succeeded for the first time in creating mutant particles which contain only monomeric RNAs without modifying any viral proteins. In terms of RNA encapsidation ability, virion density, and protein processing, the mutant particles were comparable to wild-type particles. The level of production of viral DNA by the mutant virus construct in infected cells was also comparable to that of the constructs that produced exclusively dimeric RNA, indicating that monomeric viral RNA could be the template for strand transfer. These results indicated that the RNA dimerization of HIV-1 could be separated from viral RNA packaging and was not absolutely required for RNA packaging, virion maturation, and reverse transcription.

scholarly journals Proline Residues within Spacer Peptide p1 Are Important for Human Immunodeficiency Virus Type 1 Infectivity, Protein Processing, and Genomic RNA Dimer Stability

2002 ◽  
Vol 76 (22) ◽  
pp. 11245-11253 ◽  
Author(s):  
Melissa K. Hill ◽  
Miranda Shehu-Xhilaga ◽  
Suzanne M. Crowe ◽  
Johnson Mak
Keyword(s):  
Protein Processing ◽  
Viral Infectivity ◽  
Genomic Rna ◽  
Stem Loop ◽  
Altered Protein ◽  
Immunodeficiency Virus ◽  
Rna Dimer ◽  
Hiv 1

ABSTRACT The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

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