Natural Variation in Vif: Differential Impact on APOBEC3G/3F and a Potential Role in HIV-1 Diversification

The HIV-1 Vif protein counteracts the antiviral activity exhibited by the host cytidine deaminases APOBEC3G and APOBEC3F. Here, we show that defective vif alleles can readily be found in HIV-1 isolates and infected patients. Single residue changes in the Vif protein sequence are sufficient to cause the loss of Vif-induced APOBEC3 neutralization. Interestingly, not all the detected defects lead to a complete inactivation of Vif function since some mutants retained selective neutralizing activity against APOBEC3F but not APOBEC3G or vice versa. Concordantly, independently hypermutated proviruses with distinguishable patterns of G-to-A substitution attributable to cytidine deamination induced by APOBEC3G, APOBEC3F, or both enzymes were present in individuals carrying proviruses with completely or partly defective Vif variants. Natural variation in Vif function may result in selective and partial neutralization of cytidine deaminases and thereby promote viral sequence diversification within HIV-1 infected individuals

Genetic Diversity and Phylogeny of Antagonistic Bacteria against Phytophthora nicotianae Isolated from Tobacco Rhizosphere

The genetic diversity of antagonistic bacteria from the tobacco rhizosphere was examined by BOXAIR-PCR, 16S-RFLP, 16S rRNA sequence homology and phylogenetic analysis methods. These studies revealed that 4.01% of the 6652 tested had some inhibitory activity against Phytophthora nicotianae. BOXAIR-PCR analysis revealed 35 distinct amplimers aligning at a 91% similarity level, reflecting a high degree of genotypic diversity among the antagonistic bacteria. A total of 25 16S-RFLP patterns were identified representing over 33 species from 17 different genera. Our results also found a significant amount of bacterial diversity among the antagonistic bacteria compared to other published reports. For the first time; Delftia tsuruhatensis, Stenotrophomonas maltophilia, Advenella incenata, Bacillus altitudinis, Kocuria palustris, Bacillus licheniformis, Agrobacterium tumefaciens and Myroides odoratimimus are reported to display antagonistic activity towards Phytophthora nicotianae. Furthermore, the majority (75%) of the isolates assayed for antagonistic activity were Gram-positives compared to only 25% that were Gram-negative bacteria

Infectivity and Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1 Variants Isolated during Primary Infection

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4(+) T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics

Naturally Occurring <i>vif</i> Sequence Variation

<p>The phylogenetic relationships among 79 independent <i>vif</i> sequences derived from patients (P1, P2, P3) and viral isolates (V1, V2, V3, V4) were analyzed using the Neighbor-joining method. Seven subtype B reference sequences and a consensus subtype B sequence were also included. A cluster of hypermutated <i>vif</i> sequences found in P3 is indicated. The 40 distinct protein variants selected for functional testing are identified by •. For each patient and isolate, an individual <i>vif</i> consensus sequence was generated and the percent divergence of each <i>vif</i> consensus sequence from the NL4–3 <i>vif</i> referenced is shown.</p

Phylogenetic Relationships and Hypermutation in p17MA Sequences

<div><p>(A) Neighbor joining tree representing the phylogenetic relationships among 70 independent p17MA sequences derived from patients (P1, P2, P3) and viral isolates (V1, V2, V3, V4). Additional subtype B reference sequences were also included.</p><p>(B) Graphic representation of the G-to-A changes (compared to HIV-1 NL4–3) present in p17MA sequences of P2 and P3. Analysis was performed using the HYPERMUT program [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010006#ppat-0010006-b48" target="_blank">48</a>]. All possible G-to-A substitutions in the context of the reference sequence (NL4–3) are shown with the dinucleotide context color coded (bottom four sequences).</p></div

Effect of Naturally Occurring Single Amino Acid Substitutions on NL4–3 Vif APOBEC3G and APOBEC3F Neutralization Activity

<p>The HIV-1 vector infectivity generated in the presence of APOBEC3G (A) and APOBEC3F (B) and a fixed dose of NL4–3 Vif mutant was measured. The dotted line in (B) indicates the level of infectivity observed for the vector generated in the presence of APOBEC3F and in the absence of Vif. The data represent the average infectivity as determined by at least three independent experiments, with the error bars showing the standard deviations. (C) Protein expression levels of the NL4–3 Vif mutants were determined by Western blotting of transfected 293T cell lysates. (D) Infectivity of HIV-1 vector generated in the presence of APOBEC3G (filled symbols) and APOBEC3F (open symbols) and varying levels of selected Vif mutants.</p

Analysis of <i>Gag-pol</i> Sequences Derived from LTNP and Viral Isolates

<div><p>(A) Graphic representation of the G-to-A changes (compared to HIV-1 NL4–3) present in <i>Gag-pol</i>. Analysis was performed using the HYPERMUT program [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010006#ppat-0010006-b48" target="_blank">48</a>].</p><p>(B) Quantitative summary of the observed changes. “# diff” is the number of positions at which the patient sequence differs from NL4–3. “# G-A (%)” represents the absolute number and percentage of all substitutions that are G-to-A changes. The dinucleotide context (GG, GA, GC, GT) reflects the two contiguous bases, with G-to-A mutations occurring in the first position. The numbers of differences (“# diff”) for the APOBEC3G- and APOBEC3F-induced changes in the in vitro assay are given as an average (± standard deviation) of those occurring in 10 to 15 clones of hrGFP (450 nucleotides).</p></div

Activity of Vif Variants from Patients and Viral Isolates

<div><p>(A) Infectivity of HIV-1 vector particles generated by transient transfection of 293T cells in the presence or absence of fixed amounts of APOBEC3G or APOBEC3F and the indicated amounts of HIV-1 NL4–3 Vif expression plasmids was determined, as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010006#s4" target="_blank">Material and Methods</a>. Representative results from one out of three independent experiments are depicted. Infectivity measurements were performed in duplicate assays, and the error bars represent the standard deviation of the RLU values. RLU, relative light units.</p><p>(B) APOBEC3G neutralization by Vif proteins from LTNPs (P1, P2, P3) and viral isolates (V1, V2, V3, V4). The infectivity of particles generated in the presence of APOBEC3G and each Vif protein is expressed relative to the infectivity of particles generated in the absence of APOBEC3G and Vif. Each Vif protein is identified by its source (e.g., P1, V1) and a variant number (e.g., P1–2). The data represents the average infectivity values of at least three independent experiments, with the error bars showing the standard deviations.</p><p>(C) Summary of the properties of Vif variants. Independent sequences were defined as alleles that were derived from different PCR reactions or had different nucleotide sequences. Because some changes are synonymous, not all independent sequences encode variant proteins. Vif variants with gross defects (e.g., premature stop codons) as well as those that were found to be inactive in the functional assay are designated “defective Vif.” The overall frequency of inactive Vif proteins is expressed as a percentage relative to the number of independent sequences.</p></div

Closely Related Vif Proteins Display Distinct Functional Properties

<div><p>(A) Summary of the residues implicated as causing Vif defects by comparison of functional and non-functional Vif variants. Amino acid substitutions that occurred exclusively in non-functional Vif proteins are depicted relative to the NL4–3 Vif sequence. Changes identified by * are caused by G-to-A mutations.</p><p>(B, C) Function of closely related <i>vif</i> alleles assessed by quantitation of the infectivity of particles produced in the presence of APOBEC3G (B) or APOBEC3F (C). Amino acid substitutions in the non-functional partner of the “matched” functional Vif variant are given in parentheses in (B). The dotted line in (C) indicates the level of infectivity observed for the vector generated in the presence of APOBEC3F and in the absence of Vif. The data represent the average infectivity values of at least three independent experiments, with the error bars showing the standard deviations.</p></div