Background
Human immunodeficiency virus type 1 (HIV-1) remains a global threat to public health with an estimated 36.7 million peoples living with HIV-1 (
http://www.unaids.org). The genetic diversity of HIV-1 has continued to increase, which poses an additional challenge to the treatment and prevention of HIV-1 infection [
1]. HIV-1 diversity can be attributed to low fidelity of reverse transcriptase in the process of biosynthesis of double stranded DNA, as reflected in its high rate of mutation as well as recombination between different viral strains [
2]. Therefore, a large proportion of HIV-1 strains may be defective due to the spontaneous passage of lethal mutations [
3]. In long-term non-progressors, levels of HIV-1 defectiveness have been reported to be as high as 64% in accessory genes and 41% in env V3 region [
4]. Astonishingly, defective proviruses accumulate rapidly during acute HIV-1 infection to make up over 93% of all proviruses, regardless of how early antiretroviral therapy (ART) is initiated [
5]. Defective provirus mutants may still play a role in HIV-1 pathogenesis through recombination and rescue of drug resistance phenotypes, and viral recombination may take place with defective viral forms among the quasispecies to increase viral fitness and transmission capacity [
6].
In contrast to the slow and steady change caused by mutation, recombination is a much more powerful evolutionary force. First, recombination facilitates the repair of viral genomes. Recombination can bypass Muller’s ratchet by recreating mutation free individuals from a population of mutants [
7]. Second, recombination can both create and maintain genetic diversity in a population [
8]. Third, recombination can speed adaptation by eliminating competition among beneficial mutations [
9]. Recombination is a key mechanism that facilitates the persistence of virus with latent envelope genomic fragments in the productively infected cell population [
10]. Compared with other genes of HIV-1,
env gene is undoubtedly the most variable with higher rate of mutation, deletion, and insertion [
11]. The Env glycoproteins are required when HIV-1 enters into target cells, and the diversity of the
env gene has been shown to increase continuously and peaks at the onset of AIDS [
12]. It is clear that antiviral drugs unlikely have effect on integrated viral DNA, and the efficiency of CRISPR/Cas9 gene editing technology for integrated HIV-1 DNA may also reduce because of the mutations on the defective virus [
13]. Although the defective HIV-1 occupies a considerable proportion in infections, the significance of env-defective HIV-1 mutants has not been well investigated. In this study, the evolution of superinfection of env-defective and infectious wild type HIV-1 strains in long-term in vitro passages was investigated.
Discussion
HIV-1 displays in the form of
quasispecies which is one of the hallmarks of HIV-1 infection [
21,
22]. Previous studies demonstrate that a single viral particle can lead to infection [
23,
24]. During the HIV-1 replication, the rate of nucleotide misincorporation was 3.4 × 10
− 5/base/cycle [
25]. With the high rate of mutation, the defective viruses can rapidly accumulate during acute HIV-1 infection and continue to increase as the process of the disease [
4,
5]. Even though the defective virus exists in the whole life cycle of HIV-1 infection, its effects on evolution, fitness and disease progression are rarely studied because of its non-infectious characteristic. It has been reported that morn then 1 HIV copy is found in infected spleen cells; as well, a single cell can harbor several different copies of HIV-1 NDA [
26]. Therefore, cells contain defective HIV-1 may still produce defective viral particles. Moreover, it has been revealed that HIV-1 infected cells with 5 copies of defective provirus are able to generate highly infectious viral progeny [
27]. In this study, co-transfection of the plasmids of the Env-defective virus HIV
SG3Δenv and the infectious virus HIV
NL4–3 in HEK 293 T cells resulted in a large number of recombinant progeny strains. The recombination between genes of HIV
NL4–3 and HIV
SG3Δenv increased the variety of the infectious HIV-1 strain, and the variation of HIV
NL4–3 or HIV
SG3Δenv was promoted by replacing its genome fragments with that of HIV
SG3Δenv or HIV
NL4–3, respectively.
HIV-1 superinfection can occur at any stage of the disease process despite the preexisting host immune response to the initial virus and rates of superinfection have been estimated to be close to the rates of initial infection, indicating a lack of protective immunity against newly acquired HIV-1 infection by preexisting infection [
28‐
30]. However, superinfection may be difficult to be detected when the superinfecting virus is of the same subtype as the initial virus, and recombination between these viruses is often ignored. In the study, phylogenetic analysis and bootscan breakpoint analysis were performed using HIV
NL4–3 and HIV
SG3 as parent strains, and the recombinant
env genes were firstly detected in the 11th progeny virus infected cells. By analyzing recombinant pattern and breakpoint of
env genes, it was found that the same recombinant sites appeared in different recombination patterns with one to three gene fragments replacement, implying the possibility of a second or multiple recombination. Indeed, Simon-Loriere and coworkers identified the same pattern [
31]. Due to the limited sequences amplified, the bias of recombinant hotspots might exist. However, when compared with the recombination sites identified by bootscan analysis, the results are consistent, where most of the recombination breakpoints are in the recombinant hotspots. Furthermore, recombination in the other regions of HIV-1 genome was also observed. Thus defective virus resulted from gene mutation, deletion and insertion may promote the evolution of replication-competent HIV-1 by superinfection or coinfection.
Previous studies suggest an association between HIV-1 fitness, diversity, recombination, rate of transmission, and disease progression [
32,
33]. The very fit viruses have to adapt to a given environment in order to survive. The most fit virus in an ex vivo culture suggests an increased virulence in a host. However, rapid disease progression is also related to faster extinction of this viral isolate in the human population [
34]. Ex vivo fitness of primary HIV-1 isolates typically maps to the
env gene and is largely controlled by the efficiency of host cell entry [
35]. It was shown that the recombinant Env proteins presented various infectious abilities. Compared with the HIV-1
NL4–3 strain, the fitness of all other viruses was lower, especially the rEnvV. However, the infectious ability of rEnvIII and rEnvIV was significantly increased compared to that of HIV-1
SG3. HIV-1
NL4–3 is an ex vivo fitness strain, and the nucleotide acid of
env gene is the result of an ex vivo culture adaptation. The replacement with
env gene of HIV-1
SG3ΔEnv results in a large number of mutations. Therefore, the decline of fitness of recombinant strains is predictable.
Highly active antiretroviral therapy (HAART) can effectively inhibit HIV-1 in the patients, but due to the high variation of the virus, the emergence and epidemic of drug resistant strains have become a serious problem that has to be faced. Meanwhile, the patient must take the drug for whole life in that the virus will proliferate again because of the persistence of a small reservoir of infected cells. It is reported that defective genomes were systematically detected in all patients on long-term HAART in both PBMCs and rectal tissues, and a high level of defective genomes was correlated with a small size of HIV-1 provirus DNA [
36]. Furthermore, latent HIV-1 can be activated by exosomes from cells infected defective HIV-1 [
37]. In the present study, two
env sequences and one NFLG with the characteristic inserted fragment of HIV
SG3Δenv were identified after 20 passages, suggesting that the defective HIV-1 could persist in the host and passage with the help of infectious one and served as a kind of latent HIV-1. The persistence of HIV-1 reservoir has been one of the obstacles to eradicate HIV-1 infection. The Shock/Kick and Kill strategy and CRISPR/Cas9 gene editing technology play an important role in eradicating the HIV-1 reservoir [
38‐
40]. Nevertheless, the coinfection or superinfection of defective and functional HIV-1 and high rate of recombination between them put forward a higher requirement for the elimination of the HIV-1 reservoir.
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