scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Genetic Analysis of a Unique Human Immunodeficiency Virus Type 1 (HIV-1) with a Primer Binding Site Complementary to tRNAMet Supports a Role for U5-PBS Stem-Loop RNA Structures in Initiation of HIV-1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 1818-1827 ◽  
Author(s):  
Sang-Moo Kang ◽  
Casey D. Morrow
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Rna Structures ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA3 Lys to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes tRNAMet rather than tRNA3 Lys as a primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol. 71:207–217, 1997). Comparison of RNA secondary structures of the unique sequence (U5)-primer binding site (PBS) viral RNA genome alone or complexed with tRNAMet of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to alter the U5-PBS structure and disrupt the U5-PBS-tRNAMet interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and the other predicted to destabilize contact regions of U5 with tRNAMet, reverted back to contain a PBS complementary to tRNA3 Lys. All other mutants maintained a PBS complementary to tRNAMetafter in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNAMet, maintained a PBS complementary to tRNAMet following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.

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.

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process

2000 ◽  
Vol 74 (18) ◽  
pp. 8252-8261 ◽  
Author(s):  
Hui Zhang ◽  
Roger J. Pomerantz ◽  
Geethanjali Dornadula ◽  
Yong Sun
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Binding ◽  
Viral Rna ◽  
Immunodeficiency Virus ◽  
Mrnp Complex ◽  
Rna Interaction ◽  
Vif Protein ◽  
Hiv 1

ABSTRACT Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not supportvif-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.

scholarly journals Novel Human Immunodeficiency Virus (HIV) Inhibitors That Have a Dual Mode of Anti-HIV Action

2003 ◽  
Vol 47 (10) ◽  
pp. 3109-3116 ◽  
Author(s):  
Miguel Stevens ◽  
Christophe Pannecouque ◽  
Erik De Clercq ◽  
Jan Balzarini
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protein Expression ◽  
Viral Protein ◽  
Green Fluorescence Protein Expression ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT We have found that novel pyridine oxide derivatives are inhibitors of a wide range of human immunodeficiency virus (HIV) type 1 (HIV-1) and HIV-2 strains in CEM cell cultures. Some of the compounds showed inhibitory activities against recombinant HIV-1 reverse transcriptase (RT), whereas others were totally inactive against this viral protein in vitro. Partial retention of anti-HIV-1 activity against virus strains that contain a variety of mutations characteristic of those for resistance to nonnucleoside RT inhibitors and a lack of inhibitory activity against recombinant HIV-2 RT suggested that these pyridine oxide derivatives possess a mode of antiviral action independent from HIV RT inhibition. Time-of-addition experiments revealed that these pyridine oxide derivatives interact at a postintegration step in the replication cycle of HIV. Furthermore, it was shown that these compounds are active not only in acutely HIV-1-infected cells but also in chronically HIV-infected cells. A dose-dependent inhibition of virus particle release and viral protein expression was observed upon exposure to the pyridine oxide derivatives. Finally, inhibition of HIV-1 long terminal repeat-mediated green fluorescence protein expression in quantitative transactivation bioassays indicated that the additional target of action of the pyridine oxide derivatives may be located at the level of HIV gene expression.

scholarly journals Few Mutations in the 5′ Leader Region Mediate Fitness Recovery of Debilitated Human Immunodeficiency Type 1 Viruses

2005 ◽  
Vol 79 (9) ◽  
pp. 5421-5427 ◽  
Author(s):  
Eloísa Yuste ◽  
Antonio V. Bordería ◽  
Esteban Domingo ◽  
Cecilio López-Galíndez
Keyword(s):  
Reverse Transcription ◽  
Large Population ◽  
Primer Binding Site ◽  
Leader Region ◽  
Coding Regions ◽  
Immunodeficiency Virus ◽  
Large Populations ◽  
Large Virus ◽  
Hiv 1

ABSTRACT Repeated bottleneck passages of RNA viruses result in fitness losses due to the accumulation of deleterious mutations. In contrast, repeated transfers of large virus populations result in exponential fitness increases. Human immunodeficiency virus type 1 (HIV-1) manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells. An analysis of the mutations associated with fitness loss in four debilitated clones revealed mutation frequencies in gag that were threefold higher than those in env. We now show an increase in the fitness of the debilitated HIV-1 clones by repeated passages of large populations. An analysis of the entire genomic nucleotide sequences of these populations showed that few mutations, from two to seven per clone, mediated fitness recovery. Eight of the 20 mutations affected coding regions, mainly by the introduction of nonsynonymous mutations (75%). However, most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5′ untranslated leader region of the genome, and more specifically, in the primer binding site (PBS) loop. Two of the viruses incorporated the same mutation in the primer activation signal in the PBS loop, which is critical for the tRNA3 Lys-mediated initiation of reverse transcription. Moreover, 25% of the mutations observed were reversions. This fact, together with the presence of a large proportion of nonsynonymous replacements, may disclose the operation, during large population passages, of strong positive selection for optimal HIV-1 replication, which seems to be primarily affected by binding of the tRNA to the PBS and the initiation of reverse transcription.

scholarly journals How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill?

2005 ◽  
Vol 79 (21) ◽  
pp. 13579-13586 ◽  
Author(s):  
W. David Wick ◽  
Otto O. Yang ◽  
Lawrence Corey ◽  
Steven G. Self
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.

scholarly journals Dimerization and Template Switching in the 5′ Untranslated Region between Various Subtypes of Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 77 (5) ◽  
pp. 3020-3030 ◽  
Author(s):  
Ebbe Sloth Andersen ◽  
Rienk E. Jeeninga ◽  
Christian Kroun Damgaard ◽  
Ben Berkhout ◽  
Jørgen Kjems
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Untranslated Region ◽  
Template Switching ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

scholarly journals Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

2005 ◽  
Vol 79 (16) ◽  
pp. 10356-10368 ◽  
Author(s):  
Richard Lu ◽  
Hina Z. Ghory ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Class Ii ◽  
Class I ◽  
In Vitro Assays ◽  
Mutant Proteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1E246K synthesized near-normal cDNA levels but processing of Pr55 g ag was largely inhibited in virus-producing cells. Because single-round HIV-1E246K.Luc(R-) transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1E246K. Results of complementation assays revealed that the CTD could function in trans to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently trans-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.

scholarly journals The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription

2002 ◽  
Vol 76 (5) ◽  
pp. 2329-2339 ◽  
Author(s):  
Nancy Beerens ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Sequence Motif ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA3 Lys molecule, which binds, with its 3"-terminal 18 nucleotides (nt), to a complementary sequence in the viral genome, the primer-binding site (PBS). Besides PBS-anti-PBS pairing, additional interactions between viral RNA sequences and the tRNA primer are thought to regulate the process of reverse transcription. We previously identified a novel 8-nt sequence motif in the U5 region of the HIV-1 RNA genome that is critical for tRNA3 Lys-mediated initiation of reverse transcription in vitro. This motif activates initiation from the natural tRNA3 Lys primer but is not involved in tRNA placement and was therefore termed primer activation signal (PAS). It was proposed that the PAS interacts with the anti-PAS motif in the TΨC arm of tRNA3 Lys. In this study, we analyzed several PAS-mutated viruses and performed reverse transcription assays with virion-extracted RNA-tRNA complexes. Mutation of the PAS reduced the efficiency of tRNA-primed reverse transcription. In contrast, mutations in the opposing leader sequence that trigger release of the PAS from base pairing stimulated reverse transcription. These results are similar to the reverse transcription effects observed in vitro. We also selected revertant viruses that partially overcome the reverse transcription defect of the PAS deletion mutant. Remarkably, all revertants acquired a single nucleotide substitution that does not restore the PAS sequence but that stimulates elongation of reverse transcription. These combined results indicate that the additional PAS-anti-PAS interaction is needed to assemble an initiation-competent and processive reverse transcription complex.

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