Biomedical subjects

Vinay K Pathak

Publications and source records attributed to Vinay K Pathak.

16 recordsLinked to original sources

PubMed2005

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: balance between RNase H activity and nucleotide excision.

Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of reverse transcriptase template switching, we propose that an equilibrium exists between (i) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and (ii) degradation of the RNA template by RNase H activity, leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3'-azido-3'-deoxythymidine and 2,3-didehydro-2,3-dideoxythymidine by as much as 180- and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers. These results provide insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in reverse transcriptase.

Antiviral Agents↗

PubMed2005

Mutations in the RNase H primer grip domain of murine leukemia virus reverse transcriptase decrease efficiency and accuracy of plus-strand DNA transfer.

The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either beta-galactosidase (lacZ) or green fluorescent protein (GFP) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.

Animals↗

PubMed2005

Mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor.

We previously demonstrated that human immunodeficiency virus type 1 (HIV-1) infection is nonrandom and that double infection occurs more frequently than predicted from random events. To probe the possible mechanisms for nonrandom infection, we examined the role of HIV-1 entry pathways by using viruses pseudotyped with either CCR5-tropic HIV-1 Env or vesicular stomatitis virus G protein (VSV G). These two proteins use different receptors and entry pathways. We found that regardless of the protein used, double infection occurred more frequently than random events, indicating nonrandom HIV-1 infection in both entry pathways. However, the frequency of double infection differed significantly, depending on the envelope protein. In primary CD4(+) T cells, double infection occurred most frequently when both viruses had CCR5-tropic HIV-1 Env and least frequently when the two viruses had different envelopes. These results indicated that the preference in virus entry was a significant but not the only factor contributing to nonrandom double infection. Furthermore, we demonstrated that the CD4 expression level in primary T cells affects their susceptibility to CCR5-tropic HIV-1 infection but not VSV G-pseudotyped HIV-1 infection. We have also examined infection with two viruses pseudotyped with CCR5- or CXCR4-tropic HIV-1 Env and have found that double infection occurred more frequently than random events. These results indicate that coreceptor usage is not a barrier to recombination between the two virus populations. In our previous study, we also demonstrated nonrandom double infection via dendritic cell (DC)-mediated HIV-1 transmission. To test our hypothesis that multiple HIV-1 virions are transmitted during DC-T-cell contact, we used two populations of DCs, each capturing one vector virus, and added both DC populations to T cells. We observed a decreased frequency of double infection compared with experiments in which DCs captured both viruses simultaneously. Therefore, these results support our hypothesis that multiple virions are transmitted from DCs to T cells during cell-mediated HIV-1 transmission.

CD4 Antigens↗

PubMed2004

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor.

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.

Amino Acid Sequence↗

PubMed2004

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

APOBEC-3G Deaminase↗

PubMed2004

A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion.

HIV-1 and other retroviruses occasionally undergo hypermutation, characterized by a high rate of G-to-A substitution. Recently, the human apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), first identified as CEM15, was shown to be packaged into retroviral virions and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation. This innate mechanism of resistance to retroviral infection is counteracted by the HIV-1 viral infectivity factor (Vif), which protects the virus by preventing the incorporation of APOBEC3G into virions by rapidly inducing its ubiquitination and proteasomal degradation. To gain insights into the mechanism by which Vif protects HIV-1 from APOBEC3G, we substituted several amino acids in human APOBEC3G with equivalent residues in simian APOBEC3Gs that are resistant to HIV-1 Vif and determined the effects of the mutations on HIV-1 replication in the presence and absence of Vif. We found that a single amino acid substitution mutant of human APOBEC3G (D128K) can interact with HIV-1 Vif but is not depleted from cells; thus, it inhibits HIV-1 replication in an HIV-1 Vif-resistant manner. Interestingly, rhesus macaque simian immunodeficiency virus 239 or HIV-2 Vif coexpression depleted the intracellular steady state levels of the D128K mutant and abrogated its antiviral activity, indicating that it can be a substrate for the proteasomal pathway. The HIV-1 Vif-resistant mutant APOBEC3G could provide a gene therapy approach to combat HIV-1 infection.

APOBEC-3G Deaminase↗

PubMed2004

Nonrandom HIV-1 infection and double infection via direct and cell-mediated pathways.

Cells infected with two related retroviruses can generate heterozygous virions, which are the precursors of recombinant proviruses. Although many studies have focused on the frequencies and mechanisms of retroviral recombination, little is known about the dynamics of double infection. To examine this issue, viruses generated from two HIV-1 vectors containing different markers were mixed together, and were used to infect target cells. The numbers of cells expressing none, one, or both markers were measured and were used to calculate whether double infection occurred at frequencies expected from random infection events. We found that double infection occurred significantly more frequently than predicted from random distribution; increased rates of double infection were observed in both a T cell line and primary activated CD4(+) T cells. In addition to direct virus infection, we also examined the nature of cell-mediated HIV-1 double infection. Increased double infection was observed in all experiments regardless of whether a cell line or primary human dendritic cells were used for capture and transmission of HIV-1. Therefore, our results indicate that HIV-1 double infection occurs more frequently than it would at random in both direct and cell-mediated HIV-1 infections. To our knowledge, this is the first direct evidence of nonrandom double infection in HIV-1. Frequent double HIV-1 infections in infected individuals would allow the generation of recombinant viruses that could then affect their pathogenesis and evolution.

CD4-Positive T-Lymphocytes↗

PubMed2004

Frequent dual initiation in human immunodeficiency virus-based vectors containing two primer-binding sites: a quantitative in vivo assay for function of initiation complexes.

We previously demonstrated that murine leukemia virus (MLV)-based vectors containing two primer-binding sites (PBSs) have the capacity to initiate reverse transcription more than once (Y. A. Voronin and V. K. Pathak, Virology 312:281-294, 2003). To determine whether human immunodeficiency virus (HIV)-based vectors also have the capacity to initiate reverse transcription twice, we constructed an HIV type 1 (HIV-1)-based vector containing the HIV-1 PBS, a green fluorescent protein reporter gene (GFP), and a second PBS derived from HIV-2 3' of GFP. Simultaneous initiation of reverse transcription at both the 5' HIV-1 PBS and 3' HIV-2 PBS was predicted to result in deletion of GFP. As in the MLV-based vectors, GFP was deleted in approximately 25% of all proviruses, indicating frequent dual initiation in HIV-based vectors containing two PBSs. Quantitative real-time PCR analysis of early reverse transcription products indicated that HIV-1 reverse transcriptase efficiently used the HIV-2 PBS. To investigate tRNA primer-RNA template interactions in vivo, we introduced several mutations in the HIV-2 U5 region. The effects of these mutations on the efficiency of reverse transcription initiation were measured by quantitative real-time PCR analysis of early reverse transcription products, with initiation at the HIV-1 PBS used as an internal control. Disruption of the lower and upper parts of the U5-inverted repeat stem reduced the efficiency of initiation 20- and 6-fold, respectively. In addition, disruption of the proposed interactions between viral RNA and tRNA(Lys3) thymidine-pseudouridine-cytidine and anticodon loops decreased the efficiency of initiation seven- and sixfold, respectively. These results demonstrate the relative influence of various RNA-RNA interactions on the efficiency of initiation in vivo. Furthermore, the two-PBS vector system provides a sensitive and quantitative in vivo assay for analysis of RNA-RNA and protein-RNA interactions that can influence the efficiency of reverse transcription initiation.

Base Sequence↗

PubMed2004

Antiretroviral drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase increase template-switching frequency.

Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two- to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.

Anti-HIV Agents↗

PubMed2004

Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions.

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

Base Sequence↗

PubMed2003

Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites.

Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22-29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once.

Animals↗

PubMed2003

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors.

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.

Anti-HIV Agents↗

PubMed2003

Retroviral mutation rates and reverse transcriptase fidelity.

Genetic variation in retroviral populations provides a mechanism for retroviruses to escape host immune responses and develop resistance to all known antiretroviral drugs. Retroviruses, like all RNA viruses, exhibit a high mutation rate. Polymerization errors during DNA synthesis by reverse transcriptase, which lacks a proofreading activity, is a major mechanism for generating genetic variation within retroviral populations. In this review, we summarize our current understanding of the processes that contribute to the generation of mutations in retroviruses. An overview of in vivo and in vitro studies of retroviral mutation rates determined by various fidelity assays is provided. Extensive mutational analyses of RTs are beginning to elucidate the relationship between structural determinants of RTs and fidelity of DNA synthesis. Recently, it was observed that the Y586F mutation in MLV RT results in a dramatic increase in the mutation rate in the vicinity of adenine-thymie tracts (AAAA, TTTT, and AATT), which are associated with bends in DNA. These results indicate that the template-primer duplex is a component of the polymerase active site and its structure can influence nucleotide selectivity and the mutation rate. Additionally, the results also suggest that the Y586 residue and the RNase H primer grip are structural determinants of RT that have evolved to attenuate the effects of unusual conformations of the template-primer duplex, such as bends in DNA, on fidelity of DNA synthesis.

Animals↗

PubMed2002

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Acetoacetates↗

PubMed2002

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine-thymine tracts.

Using in vivo fidelity assays in which bacterial beta-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate approximately 5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine-thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template-primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adenine-thymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template-primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template-primer conformation at the polymerase active site and that the MLV Y586 residue and template-primer conformation are important determinants of RT fidelity.

Amino Acid Sequence↗

PubMed2002

Zinc finger domain of murine leukemia virus nucleocapsid protein enhances the rate of viral DNA synthesis in vivo.

In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

Amino Acid Motifs↗