HIV-1 Gag-Pol Sequences from Ugandan Early Infections Reveal Sequence Variants Associated with Elevated Replication Capacity

Abstract

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.

Keywords: Gag-Pol; HIV-1; Uganda; protein domains; recombinant.

Figure 1

HIV-1 breakpoint map showing the recombination patterns across the 13 recombinants. This was generated using the jpHMM website and recombinant HIV-1 drawing tool from the LANL website as described in Materials and Methods. The key to colors in the figure: red as A1, light green as D, brown as C, dark green as G and light blue as 01_AE.

Figure 2

Histogram of VRC values across the 32 samples. The orange dashed line indicates a smoothed kernel density estimation (as implemented in Seaborn) of VRC values.

Figure 3

(A) There was no difference between the subtype classification of A1, D or the recombinants and the time taken for CD4⁺ T cell counts to decline to 350 cells/µL over the first 5.5 years of infection. (B) There was no difference between the subtypes and set point viral load.

Figure 4

(A) Protein domains with variation across a query set of 32 HIV-1 Gag-Pol sequences. The HIV-1 Gag-Pol region is depicted, with open reading frames for gag (light blue) and pol (gray) indicated. The positions of HIV-1 Pfam domains with low variation (green) and the seven higher variation domains used here are colored in orange. (B) Variance of domain bit scores across the set of 32 query sequences.

Figure 5

VRC, total domain scores and Gag-p6 domain bit scores by subtype. Scores for the 32 sequences were plotted as a function of the subtype category. For each panel, the median value is indicated by a horizontal line within the box, the top and bottom of the box indicate the interquartile range, individual values are indicated by red markers. Intergroup p values (Welch’s t-test) showed that all pairs were different (A1_VRC, D_VRC, p value = 0.0017; A1_VRC, A1D_VRC, p value = 0.0010; A1_VRC, Other_Recomb_VRC, p value = 0.0008; D_VRC, A1D_VRC, p value = 0.0281; D_VRC, Other_Recomb_VRC, p value = 0.0007; A1D_VRC, Other_Recomb_VRC, p value = 0.0151). (A) VRC vs. subtype, (B) Total domain bit-score vs subtypes, (C) Gag-P6 bitscore vs subtype.

Figure 6

Protein changes in Gag-P6 region. The amino acid sequence of the Gag-P6 domains from the 32 sequences were aligned and a sequence logo was generated using Weblogo3 [39]. Amino acids are indicated by a single letter code with the height of each letter stack indicating conservation at that position (measured in entropy bits, see [39]) and the height of the letter within the stack indicating the relative frequency of the amino acid at that position. Amino acids found only in the genomes with VRC ≤ 0.4 are indicated in red.

Figure 7

Comparing Gag-P6 bit scores for early infection sequences and all available HIV-1 genomes. All available HIV-1 genome sequences were retrieved from GenBank and classified by subtype using the KAMERIS tool [37]. Gag-P6 Pfam domains were identified and bit scores were gathered. The plot shows standard boxplots of the bit scores, stratified by the 25 HIV-1 subtypes identified in the set of genomes, with first interquartile range indicated by a colored box and the median value for each group indicated by a horizontal line. The Gag-P6 bit scores for the early sequences reported here are shown with red markers in their corresponding subtype. The counts of genomes by subtype were subtype B:7186, C:1750, 01_AE:1092, A1:414, 02_AG:244, BF1:225, G:178, 01B:168, BC:161, U:122, A1D:108, A1C:105, D:101, A6:98, F1:93, 02A1:83, CD:77, 01BC:72, O:71, BF:56, 07_BC:52, 08_BC:39, 11_cpx:29, A1CD:28, 35_AD:21, total: 12,573.