Host Restriction Factors APOBEC3G/3F and Other Interferon-Related Gene Expressions Affect Early HIV-1 Infection in Northern Pig-Tailed Macaque (Macaca leonina)

Abstract

The northern pig-tailed macaques (NPMs) lack TRIM5α, an antiviral restriction factor, and instead have TRIM5-CypA. In our previous study, we demonstrated that HIV-1_NL4-3 successfully infected NPMs and formed a long-term viral reservoir in vivo. However, the HIV-1-infected NPMs showed relatively high viremia in the first 6 weeks of infection, which declined thereafter suggesting that HIV-1 _NL4-3 infection in these animals was only partly permissive. To optimize HIV-1 infection in NPMs therefore, we generated HIV-1_NL4-R3A and stHIV-1sv, and infected NPMs with these viruses. HIV-1_NL4-R3A and stHIV-1sv can replicate persistently in NPMs during 41 weeks of acute infection stage. Compared to the HIV-1_NL4-R3A, stHIV-1sv showed a notably higher level of replication, and the NPMs infected with the latter induced a more robust neutralizing antibody but a weaker cellular immune response. In addition, IFN-I signaling was significantly up-regulated with the viral replication, and was higher in the stHIV-1sv infected macaques. Consequently, the sequences of pro-viral env showed fewer G-A hyper-mutations in stHIV-1sv, suggesting that vif gene of SIV could antagonize the antiviral effects of APOBEC3 proteins in NPMs. Taken together, NPMs infected with HIV-1_NL4-R3A and stHIV-1sv show distinct virological and immunological features. Furthermore, interferon-related gene expression might play a role in controlling primary HIV-1_NL4-R3A and stHIV-1sv replication in NPMs. This result suggests NPM is a potential HIV/AIDS animal model.

Keywords: APOBEC3; HIV-1NL4-R3A; IFN-I signaling; neutralizing antibodies; northern pig-tailed macaques; stHIV-1sv.

Figure 1

HIV-1_NL4−R3A and stHIV-1sv replicated more robustly than HIV-1_NL4.3 in PBMCs of northern pig-tailed macaques. (A) The genomic structures of HIV-1_NL4−R3A, and stHIV-1sv in comparison to that of HIV-1_NL4−3. (B) Replication of HIV-1_NL4−3, HIV-1_NL4−R3A and stHIV-1sv in PBMCs of NPMs. 1 × 10⁵ PBMCs from 2 healthy donors of NPMs were infected with these 3 viruses at an MOI of 0.01, replication was monitored by determining P24 concentration in supernatant at 3-day intervals post-infection.

Figure 2

The primary infection of northern pig-tailed macaques with HIV-1_NL4−R3A and stHIV-1sv. 4 NPMs were infected with HIV-1_NL4−R3A and stHIV-1sv, respectively. (A) Plasma viral load, (B) B cell counts, (C) CD4+ T cell counts, (D) CD8+ T cell counts, (E) the ratio of CD4+ to CD8+ T cells in peripheral blood in the primary infection of 28 weeks were measured. P-value differences between HIV-1_NL4−R3A and stHIV-1sv infected groups were calculated by two-way analysis of variance.

Figure 3

PBMC associated viral DNAs and RNAs in HIV-1_NL4−R3A and stHIV-1sv infected NPMs. (A) PBMC associated viral DNAs, (B) PBMC associated viral RNAs, (C) the proportion of viral RNAs to viral DNAs. P value differences between HIV-1_NL4−R3A and stHIV-1sv infected groups were calculated by two-way analysis of variance.

Figure 4

Antibody and Cotoxic lymphocyte(CTL) responses to the primary infection of northern pig-tailed macaques with HIV-1_NL4−R3A and stHIV-1sv. (A) Antibody responses to HIV-1_NL4−R3A and stHIV-1sv infection in NPMs were detected by western blotting analysis, the plasma at 4, 8, and 12 weeks postinfection (wpi) were used. (B) HIV-1 special neutralizing antibodies against these two viruses were measured respectively at 4, 12, 18 wpi. The inactivated plasma mixed with corresponding virus was added into Tzm-bl and the 50% reductions of virus infectivity (IC50) were calculated. (C) Cotoxic lymphocyte (CTL) responses to these two viruses were determined at 4 and 7 wpi. P-values were calculated by t-test.

Figure 5

The expression of IFN-related genes during primary infection of HIV-1_NL4−R3A and stHIV-1sv. (A) The hotmap of IFN-related genes expression. 22 genes were significantly induced by HIV-1_NL4−R3A and stHIV-1sv infection (wpi = 1, 2, 3, 4, 6, 12, and 24) and differentially expressed in the HIV-1_NL4−R3A (n = 4) and stHIV-1sv (n = 4) infected macaques. Clusters were performed on the average of log2 ratios of mRNA expression relative to the basal levels before infection by the RT-PCR-based ΔΔCt method. The progressive decreases or increases in mean log2-fold-change were represented by the changes of blue to red colors. (B) The dynamic expression of some selected genes involved in IFNα2, IRF7, and restriction factors MX2, Tetherin, APOBEC3G and APOBEC3F. P-value differences between HIV-1_NL4−R3A and stHIV-1sv infected groups were calculated by two-way analysis of variance.

Figure 6

APOBEC3-induced HIV-1 hypermutation may contribute to the inhibition of virus replication during primary infection. Samples from HIV-1_NL4−R3A and stHIV-1sv infected 4 NPMs, respectively. The weeks post-infection were showed at the right side. Number in the table displayed the amount of hypermutation. There were more mutations caused by APOBEC3 family in HIV-1_NL4−R3A infected macaques than stHIV-1sv. The colors represented as: Red tick mark, GG>AG mutation (APOBEC3G pattern); Cyan tick mark, GA>AA mutation (APOBEC3F pattern); Green tick mark, GC>AC mutation; Magenta tick mark, GT>AT mutation; all other mutations; Yellow tick mark, deletion.