A New Generation

##Generation of a monkey-tropic human immunodeficiency virus type 1 carrying env from a CCR5-tropic subtype C clinical isolate

[source] 2ahUKEwjO06CIwcfjAhUVfH0KHT1oDPkQ_AUoAXoECAwQAQ&biw=360&bih=247

Abstract:Several derivatives of human immunodeficiency virus type 1 (HIV-1) that evade macaque restriction factors and establish infection in pig-tailed macaques (PtMs) have been described. These monkey-tropic HIV-1s utilize CXCR4 as a co-receptor that differs from CCR5 used by most currently circulating HIV-1 strains. We generated a new monkey-tropic HIV-1 carrying env from a CCR5-tropic subtype C HIV-1 clinical isolate. Using intracellular homologous recombination, we generated an uncloned chimeric virus consisting of at least seven types of recombination breakpoints in the region between vpr and env. The virus increased its replication capacity while maintaining CCR5 tropism after in vitro passage in PtM primary lymphocytes. PtM infection with the adapted virus exhibited high peak viremia levels in plasma while the virus was undetectable at 12–16 weeks. This virus serves as starting point for generating a pathogenic monkey-tropic HIV-1 with CCR5-tropic subtype C env, perhaps through serial passage in macaques.
[source] https://www.sciencedirect.com/science/article/pii/S0042682214001883
Results
Generation of a new HIV-1mt carrying CCR5-tropic subtype C env through IHR
We employed IHR to generate recombinant viruses (Fujita et al., 2013). First, we prepared DNA fragments by polymerase chain reaction (PCR) amplification of a region spanning the 5′ long terminal repeat (LTR) to upstream of the V1/V2 region in env (nucleotide positions 1–6784 based on HXB2 numbering; accession number: K03455) using the plasmid DNA template encoding the full-length NL-DT5R proviral genome (fragment I in Fig. 1A). This fragment encodes a CypA-binding motif derived from the corresponding sequence of SIVmac239 to evade restriction from macaque TRIM5α, and the entire SIVmac239 vif gene to counteract the macaque APOBEC3G. Second, a region spanning the vpr gene to the R region of the 3′ LTR (nucleotide positions 5558–9625 based on HXB2 numbering) was amplified from the HIV-1 97ZA012 strain (fragment II in Fig. 1B). To increase the possibility to obtain a virus that can replicate in monkeys well, we thought that it was better to generate swarm viruses having variation without cloning. Resultant recombinant virus might fail to replicate normally if recombination occurred between fragments I and II that resulted in the 5′ LTR of subtype B and the 3′ LTR of subtype C. The discordance of the 3′ and 5′ LTR may disrupt successful translocation of the minus strand strong stop DNA to the plus strand genomic RNA during reverse transcription (Goff, 2007). To match the sequence of the 3′ LTR to that of the 5′ LTR, we prepared a third DNA fragment encoding a region spanning the 5′ LTR to the middle of gag (nucleotide positions 1–1433 based on HXB2 numbering) from the proviral DNA extracted from HIV-1 97ZA012-infected cells (fragment III in Fig. 1B). Fragments I and II had an overlapping region between the initiation of vpr to upstream of the env V1/V2 region, and fragments I and III had an overlapping region between the 5′ LTR to upstream of the CypA-binding site.

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