A Cell-Free Stock of Simian–Human Immunodeficiency Virus That Causes AIDS in Pig-Tailed Macaques Has a Limited Number of Amino Acid Substitutions in Both SIVmacand HIV-1 Regions of the Genome and Has Altered Cytotropism

ABSTRACT The compound 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

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Effects of the Δ67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

Journal of Virology ◽

10.1128/jvi.78.18.9987-9997.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9987-9997 ◽

Cited By ~ 19

Author(s):

Paul L. Boyer ◽

Tomozumi Imamichi ◽

Stefan G. Sarafianos ◽

Edward Arnold ◽

Stephen H. Hughes

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Amino Acid Substitutions ◽

Wild Type ◽

Coding Region ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Long-term use of combination therapy against human immunodeficiency virus type (HIV-1) provides strong selective pressure on the virus, and HIV-1 variants that are resistant to multiple inhibitors have been isolated. HIV-1 variants containing amino acid substitutions within the coding region of HIV-1 reverse transcriptase (RT), such as the 3′-azido-3′-deoxythymidine (AZT)-resistant variant AZT-R (M41L/D67N/K70R/T215Y/K219Q) and a variant containing an insertion in the fingers domain (S69SGR70/T215Y), are resistant to the nucleoside RT inhibitor (NRTI) AZT because of an increase in the level of excision of AZT monophosphate (AZTMP) from the primer. While rare, variants have also been isolated which contain deletions in the RT coding region. One such virus, described by Imamichi et al. (J. Virol 74:10958-10964, 2000; J. Virol. 74:1023-1028, 2000; J. Virol. 75:3988-3992, 2001), contains numerous amino acid substitutions and a deletion of codon 67, which we have designated the Δ67 complex of mutations. We have expressed and purified HIV-1 RT containing these mutations. We compared the polymerase and pyrophosphorolysis (excision) activity of an RT with the Δ67 complex of mutations to wild-type RT and the two other AZT-resistant variants described above. All of the AZT-resistant variants we tested excise AZTMP and 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA [tenofovir]) from the end of a primer more efficiently than wild-type RT. Although the variant RTs excised d4TMP less efficiently than AZTMP and PMPA, they were able to excise d4TMP more efficiently than wild-type RT. HIV-1 RT containing the Δ67 complex of mutations was not able to excise as broad a range of NRTIs as the fingers insertion variant SSGR/T215Y, but it was able to polymerize efficiently with low concentrations of deoxynucleoside triphosphates and seems to be able to excise AZTMP and PMPA at lower ATP concentrations than AZT-R or SSGR/T215Y, suggesting that a virus containing the Δ67 complex of mutations would replicate reasonably well in quiescent cells, even in the presence of AZT.

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S-1153 Inhibits Replication of Known Drug-Resistant Strains of Human Immunodeficiency Virus Type 1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.6.1340 ◽

1998 ◽

Vol 42 (6) ◽

pp. 1340-1345 ◽

Cited By ~ 82

Author(s):

Tamio Fujiwara ◽

Akihiko Sato ◽

Mohamed El-Farrash ◽

Shigeru Miki ◽

Kenji Abe ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Reverse Transcriptase ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Reverse Transcriptase Inhibitors ◽

Human T Cell ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT S-1153 is a new imidazole compound that inhibits human immunodeficiency virus (HIV) type 1 (HIV-1) replication by acting as a nonnucleoside reverse transcriptase inhibitor (NNRTI). This compound inhibits replication of HIV-1 strains that are resistant to nucleoside and nonnucleoside reverse transcriptase inhibitors. S-1153 has a 50% effective concentration in the range of 0.3 to 7 ng/ml for strains with single amino acid substitutions that cause NNRTI resistance, including the Y181C mutant, and also has potent activity against clinical isolates. The emergence of S-1153-resistant variants is slower than that for nevirapine, and S-1153-resistant variants contained at least two amino acid substitutions, including F227L or L234I. S-1153-resistant variants are still sensitive to the nucleoside reverse transcriptase inhibitors zidovudine (AZT) and lamivudine. In a mouse and MT-4 (human T-cell line) in vivo HIV replication model, S-1153 and AZT administered orally showed a marked synergy for the inhibition of HIV-1 replication. S-1153 shows a significant accumulation in lymph nodes, where most HIV-1 infection is thought to occur. S-1153 may be an appropriate candidate for two- to three-drug combination therapy for HIV infection.

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In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.6.1313 ◽

1997 ◽

Vol 41 (6) ◽

pp. 1313-1318 ◽

Cited By ~ 23

Author(s):

M Tanaka ◽

R V Srinivas ◽

T Ueno ◽

M F Kavlick ◽

F K Hui ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Amino Acid Substitutions ◽

Infectious Clones ◽

Immunodeficiency Virus ◽

Hiv 1

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

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Construction of a Human Immunodeficiency Virus Type 1 (HIV-1) Library Containing Random Combinations of Amino Acid Substitutions in the HIV-1 Protease due to Resistance by Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.76.6.3031-3037.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 3031-3037 ◽

Cited By ~ 6

Author(s):

Keisuke Yusa ◽

Wei Song ◽

Matthias Bartelmann ◽

Shinji Harada

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Amino Acid Substitutions ◽

In Vitro System ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 × 105) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 μM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.

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Amino Acid Insertions near Gag Cleavage Sites Restore the Otherwise Compromised Replication of Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.78.21.12030-12040.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 12030-12040 ◽

Cited By ~ 43

Author(s):

Sadahiro Tamiya ◽

Sek Mardy ◽

Mark F. Kavlick ◽

Kazuhisa Yoshimura ◽

Hiroaki Mistuya

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Amino Acid Substitutions ◽

Cleavage Sites ◽

Wild Type ◽

Gag Proteins ◽

Infectious Clones ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.

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Gln145Met/Leu Changes in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Resistance to Nucleoside and Nonnucleoside Analogs and Impair Virus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4611-4617.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4611-4617 ◽

Cited By ~ 12

Author(s):

Stefania Paolucci ◽

Fausto Baldanti ◽

Giovanni Maga ◽

Reynel Cancio ◽

Maurizio Zazzi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Multidrug Resistance ◽

Amino Acid ◽

Reverse Transcriptase ◽

Virus Replication ◽

Amino Acid Substitutions ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.

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Identification of Biased Amino Acid Substitution Patterns in Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.73.7.6197-6202.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 6197-6202 ◽

Cited By ~ 44

Author(s):

Robert W. Shafer ◽

Phillip Hsu ◽

Amy K. Patick ◽

Charles Craig ◽

Volker Brendel

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Amino Acid Substitution ◽

Sampling Error ◽

Amino Acid Substitutions ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) amino acid substitutions observed during antiretroviral drug therapy may be caused by drug selection, non-drug-related evolution, or sampling error introduced by the sequencing process. We analyzed HIV-1 sequences from 371 untreated patients and from 178 patients receiving a single protease inhibitor. Amino acid substitution patterns during treatment were compared with inferred substitution patterns arising evolutionarily without treatment. Our results suggest that most treatment-associated amino acid substitutions are caused by selective drug pressure, including substitutions not previously associated with drug resistance.

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Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

Journal of Virology ◽

10.1128/jvi.73.1.19-28.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 19-28 ◽

Cited By ~ 35

Author(s):

David E. Ott ◽

Elena N. Chertova ◽

Laura K. Busch ◽

Lori V. Coren ◽

Tracy D. Gagliardi ◽

...

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hydrophobic Tail ◽

Carboxyl Terminal ◽

Hiv 1

ABSTRACT The p6Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6Gag between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6Gag proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

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Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.01213-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 9875-9889 ◽

Cited By ~ 10

Author(s):

Elodie Beaumont ◽

Daniela Vendrame ◽

Bernard Verrier ◽

Emmanuelle Roch ◽

François Biron ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Matrix Protein ◽

Immunodeficiency Virus ◽

The Matrix ◽

Hiv 1

ABSTRACT Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs). The strong conservation of CT length in primary isolates of HIV-1 suggests that this factor plays a key role in viral replication and persistence in infected patients. However, we report here the emergence and dominance of a primary HIV-1 variant carrying a natural 20-amino-acid truncation of the CT in vivo. We demonstrated that this truncation was deleterious for viral replication in cell culture. We then identified a compensatory amino acid substitution in the matrix protein that reversed the negative effects of CT truncation. The loss or rescue of infectivity depended on the level of Env incorporation into virus particles. Interestingly, we found that a virus mutant with defective Env incorporation was able to spread by cell-to-cell transfer. The effects on viral infectivity of compensation between the CT and the matrix protein have been suggested by in vitro studies based on T-cell laboratory-adapted virus mutants, but we provide here the first demonstration of the natural occurrence of similar mechanisms in an infected patient. Our findings provide insight into the potential of HIV-1 to evolve in vivo and its ability to overcome major structural alterations.

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