Journal of AIDS and Immune Research

Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome?

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Published Date: March 02, 2015

Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? 

   
Weining Han1, Yuejin Li2, Bernard S. Bagaya3, Meijuan Tian2, Mastooreh Chamanian3, Chuanwu Zhu4, Jie Shen1, Yong Gao2,3*         

1Suzhou Center for Disease Control and Prevention, Suzhou, China
2Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave, Cleveland, Ohio 44106, USA
3Department  of  Molecular  Biology  and  Microbiology,  School  of  Medicine,  Case  Western  Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA
4The Fifth People’s Hospital, Suzhou, China

*Corresponding author: Yong Gao, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave, Cleveland, Ohio 44106, USA, E-mail: Yong.gao@case.edu  

Citation: Han W , Li Y, Bagaya BS, Tian M, Chamanian M, et al. (2015) Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome? J Aids Imm Res 1(1): 101.

 

Abstract 


Although the process of reverse transcription is well elucidated, it remains unclear if viral core disruption provides a more cellular or viral milieu for HIV-1 reverse transcription. We have devised a method to require mixing of viral cores or core constituents to produce infectious progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each other and when together can complete viral reverse transcription. Only the heterodiploid virus containing both the nfl and cplt_R/U5/gag/Δpol sgRNAs can complete reverse transcription and propagate infectious virus upon de novo infection. Dual exposure of U87.CD4.CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/Δpol virus particles did not result in productive virus infection. On the other hand, in early endosomes, the HIV-1 sgRNAs released from viral cores can retain function and complete the reverse transcription and result in productive infection. These findings confirm the assumptions that, in natural infection, HIV-1 cores, and likely other retrovirus cores, remain largely intact and do not mix/fuse in the cytoplasm during the reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA (produced through transfection) could not help the complementary sgRNA in the viral core to complement the reverse transcription process.

Keywords: Subgenomic RNAs; Immunodeficiency; reverse transcription 

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Introduction 


HIV-1 enters target cell via specific binding of viral envelope glycoprotein gp120 with cell receptors CD4 and CCR5 (or CXCR4) [1]. Entry can occur on the cell membrane with or without endocytosis. Reverse transcriptase is triggered upon expulsion of the core into the cytoplasm after viral-host membrane fusion. Studies suggest that the core is partly disrupted to permit influx of dNTPs, to promote reverse transcription, and to eventually form the pre-integration complex which is actively transported to the nucleus through a nuclear pore [2-4].

The key reverse transcription (RT) event usually lasts for 8-12h and is a very complicated and highly ordered process. In addition to viral reverse transcriptase, the process may involve the participation of cellular elements such as the cytoskeleton [5] and other viral proteins, such as Tat and Vif [6]. Although the process of reverse transcription is well characterized, the role of the viral capsid during reverse transcription remains unclear [7,8]. Previous studies [9,10] showed that uncoating occurs shortly after the membrane fusion event. This uncoating is necessary for the formation of the reverse transcription complex (RTC) [11-14]. However, the conical core structure is unstable and very sensitive to even the mildest detergent treatment used in these density gradient analyses [15], thus, it is quite possible that the loose structure observed in purified cores does not reflect a more minimal dissociation of their components in the cytoplasm after virus entry and early reverse transcription events. Direct observation by scanning electron microscopy (SEM) indicates that uncoating does not occur as an immediate post-fusion event, but rather during late synthesis of proviral DNA synthesis and transport of the pre-integration complex (PIC) to the nuclear pore [16]. In support of this hypothesis, we discovered that siRNAs associated with the RISC complex could not degrade HIV-1 genomic RNA following virus entry suggesting inability to penetrate the core. However, core dissolution was sufficiently increased by the addition of rhesus TRIM5α such that the siRNA-RISC complex could then degrade genomic RNA soon after virus entry [17]. This study again suggests that, even though the viral core possibly becomes loose to facilitate viral reverse transcription, but is still able to prevent the access of big host molecules in the host cells.

Reverse transcription is initiated from the host tRNALys3, acting as a primer, and annealed to the primer binding site (PBS) located downstream of the 5?-LTR of the HIV-1 RNA genome. The first strong stop of (-) strand DNA synthesis occurs following transcription of the U5 and R (repeat) regions, which then triggers RNase H degradation of this RNA template, freeing the (-) strand strong stop DNA to pair with the R region on the opposite end of the same genomic RNA template (intrastrand) or the other genomic RNA template (interstrand) within the core. The preference for an intra- versus inter-strand switch has been debated for over 30 years. Our recent study had separated the HIV-1 genome into two distinct RNA species, the first subgenomic lacking the 5’ R and U5 regions (i.e. near full length or nfl sgRNA) and second only containing 5’ R, U5, PBS, and necessary RNA packaging elements (i.e. complementing or cplt RNA). The nfl sgRNA can also serve as mRNA to translate the entire HIV-1 proteome in the correct stoichiometry, which subsequently results in virus particle production. However, only heterodiploid HIV-1 particles containing both the nfl and cplt sgRNAs can complete reverse transcription upon de novo infection (Figure 1) [18]. It is important to note that the cplt sgRNA only acts as a template for (-) strand strong-stop DNA synthesis which can then jump onto to the nfl sgRNA to complete proviral DNA synthesis and virus propagation. This bipartite genome system provided a unique method to explore HIV-1 core stability and possible core mixing/fusing within the cytoplasm via dual infections or transfection with virus particles harboring only one or the other sgRNAs. We could also express these sgRNAs in the cytoplasm and look for diffusion into virus core. Possible core mixing was assessed by completion of reverse transcription and virus propagation. Theoretically, if the substantial core dissociation is required for HIV reverse transcription, then two HIV-1 cores in the cells or free sgRNAs may mix to mediate complete reverse transcription and lead to virus propagation. Core mixing/fusing could even be promoted by the notion that HIV-1 core are transported to the microtubule complex and subsequent migrated along tubulin to the nuclear pore [19]. However, if core structure is maintained and cores cannot mix during virus entry and early reverse transcription events, then these two virus particles would be incapable of completing reverse transcription and mediating a productive infection. In relation to this hypothesis, we set up several different conditions for co-exposing cells to the virus particle containing the nfl and/or cplt sgRNAs in association to high levels of mixing of these subgenomic RNAs. 

Figure 1: HIV-1 production system with two complementary HIV-1 RNA genomes. (A) Two plasmids, pREC_nfl_NL4-3, which transcribes near full length of HIV-1 RNA (lack of 5?LTR) and produces the full cadre of HIV-1 proteins, and pREC_cplt_R/U5/gag/Δpol, which contains R, U5, PBS, packaging sequence, gag, and partial pol genes. (B) Virus from co-transfection of 293T cells containing cplt_R/U5/gag/Δpol and nfl HIV-1 sgRNAs can complete the reverse transcription process in a manner analogous to the intrastrand model of retroviral reverse transcription

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Methods 

 

Plasmids
The plasmids pREC_nfl_NL4-3 (containing all of the HIV-1 sequences except the R and U5 sequences) and pREC_cplt_R/U5/gag/Δpol (containing R, U5, PBS, and gag and partial pol sequences) were constructed in our lab [18]. The packaging plasmid ΔR8.91 (encoding HIV gag and pol proteins), plasmid pREC_HIV env (encoding HIV-1 envelope glycoprotein), and plasmid pMD.G (encoding vesicular stomatitis virus (VSV) G envelope glycoprotein), were kindly provided by Dr. Stanton L. Gerson [20].

Cell culture
The U87.CD4.CXCR4 cell line?human glioma cell line expressing CD4 and CXCR4 receptors) was obtained from the AIDS Research and Reference Reagent Program and maintained in Dulbecco’s modified Eagle medium (DMEM) with 15% FBS supplemented with 100U of penicillin, 100mg of streptomycin, 300μg of G418 (Life Technologies, Inc.), and 1μg of puromycin per ml. 293T cells were obtained from the American Type Culture Collection and were grown in DMEM with 10% FBS supplemented with 100U of penicillin and 100μg of streptomycin per ml. Both cell lines were grown at 37°C in 5% CO2.


Pseudotyped virus production and virtual TCID50
Pseudotyped viruses were produced as described by Zielske et al [20]. Using Lipofectamine 2000 , 293T cells were triple transfected with pREC_cplt_R/U5/gag/Δpol (or pREC_nfl_NL4-3), pSM-WT (or pMD.G), and ?R8.91 at a mass ratio of 3:1:3 to produce: pseudoviruses containing HIV-1 cplt_R/U5/gag/Δpol sgRNA wrapped with HIV-1 envelope (Virus #1) or VSV-G envelope (Virus #2), pseudoviruses containing HIV-1 nfl sgRNA with HIV-1 envelope (Virus #3), pseudoviruses containing HIV-1 nfl sgRNA with both HIV-1 and VSV-G envelopes (Virus #4), and pseudoviruses containing both sgRNAs wrapped with HIV-1 envelope (Virus #5) (Figure 2). Forty-eight hours after transfection, virus-containing cell-free supernatants were collected and stored at -80°C for further use. 

Figure 2: Schematics of construction of different pseudotyped viruses. 293T cells were transfected with pREC_cplt_R/U5/gag/Δpol, pREC_HIV_env (or pMD.G), and ?R8.91 at a mass ratio of 3:1:3 to produce: pseudoviruses containing HIV-1 cplt_R/U5/gag/Δpol sgRNA wrapped with HIV-1 envelope (Virus #1) or VSV-G envelope (Virus #2), with pREC_nfl_NL4-3 alone to produce pseudovirus containing HIV-1 nfl sgRNA with HIV-1 envelope (Virus #3), with pMD.G and pREC_nfl_NL4-3 to produce pseudoviruses containing HIV-1 nfl sgRNA with both HIV-1 and VSV-G envelopes (Virus #4), and with pREC_cplt_R/U5/gag/Δpol and pREC_nfl_NL4-3 to produce pseudoviruses containing both HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs wrapped with HIV-1 envelope (Virus #5).

 

Produced pseudotyped viruses were titrated via virtual TCID50 assay as previously described [21]. Briefly, the sampled viruses were prepared using serial four-fold dilutions, along with one reference virus with a known TCID50. 10μl of each dilution of each virus was transferred to the round bottom 96-well plate (in triplicate) for the subsequent RT assay. The obtained RT value of the reference virus was plotted versus infectious units (IU) at each dilution in the linear range, and an equation was generated based on this relationship. The virtual TCID50 of the sample virus was calculated by inserting the RT value into the equation.

Figure 3: Production and titration of five pseudotyped viruses. (A) Real-time RT-PCR detection of cplt_R/U5/gag/Δpol and nlf sgRNAs in virus particle #1, #2, #3, #4, and #5 produced from transfected 293T cells. (B) RT assay detection of reverse transcriptase activity in the five different virus particles. 

 

Transfection and infection
Twenty four hours after being plated (3×104 cells per well in 48-well plates), the U87.CD4.CXCR4 cells were transfected with certain plasmids, i.e. pREC_cplt_R/U5/gag/Δpol or pREC_nfl_NL4-3. 24 hours post-transfection, the cells were exposed to different pseudoviruses to create different settings for viral sgRNAs (i.e. in the cytoplasm and/or viral core through different entry pathways). For entrance through the HIV envelope pathway, U87.CD4.CXCR4 cells were transfected with pREC_cplt_R/U5/gag/Δpol and infected with Virus #3, transfected with pREC_nfl_NL4-3 and infected with Virus #1, or infected with virus #5 only (Figure 5A). For entrance through the VSV-G envelope pathway, U87.CD4.CXCR4 cells were transfected with pREC_cplt_R/U5/gag/Δpol and infected with Virus #4, or transfected with pREC_nfl_NL4-3 and infected with Virus #2 (Figure 6A). Viral infectivity was monitored at different time points by RT assay post-infection. Co-infections with virus #1and #3, or #2 and #4 (MOI 10) were also performed (Figure 4A). 

Figure 4: Detection of HIV-1 production in dual infection where the complementary viral subgenomes were located in different capsids. (A) Three different coexisting patterns of the complementary HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs: sgRNAs are in different viral cores wrapped with HIV envelopes (Condtion I); sgRNAs are in different viral cores wrapped with VSV-G envelopes (Condition II); and sgRNAs are in same viral core wrapped with HIV envelopes as a positive control. (B) Real-time RT-PCR detection of sgRNAs in U87.CD4.CXCR4 cells infected by viruses in different combinations. (C) RT assay detection of HIV-1 production post-infection of U87.CD4.CXCR4 cells.

 

To assure that the two different viral cores entered the same cell, so that the cplt_R/U5/gag/Δpol and nfl_NL4-3 sgRNAs could stay together to complement one another during the reverse transcription process, we fused the two virus particles with liposome (Effectene, Qiagen). Virus #1 and #3, as well as Virus #2 and #4, were mixed together with equal amount of viruses, and then incubated with 7μl of Effectene per ml of virus at room temperature for 6 hours. The fused virus particles with HIV-1 env or VSV-G env were then used to infect U87.CD.CXCR4 cells, and these were monitored for virus production at different time points (i.e. Day 3, 5, 10, 14, 17, 24 post-infection) (Figure 7).

Figure 5: Detection of HIV-1 production in HIV env pathway where the complementary viral subgenomes were separated in cytoplasm and capsid. (A) Three different coexisting patterns of HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs: cplt_R/U5/gag/Δpol sgRNA produced in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_ cplt_R/U5/gag/Δpol vector, and nfl sgRNA located in viral core through HIV-1 envelope-mediated entry (Condition III); nfl sgRNA produced in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_nfl_NL4-3 vector (Condition IV), and cplt_R/U5/gag/Δpol sgRNA located in viral core through HIV-1 envelope-mediated entry; and both sgRNAs are in same viral core wrapped with HIV envelopes. (B) Real-time RT-PCR detection of sgRNAs in U87.CD4.CXCR4 cells infected/transfected in different combinations. (C) RT assay detection of HIV-1 virus production post-infection/transfection of U87.CD4.CXCR4 cells.

 

Real-time PCR analysis of viral RNA in transfected/infected cells
Celluar RNA was extracted from U87.CD4.CXCR4 cells infected with different virus particles and/or transfected with various vectors using the RNeasy Mini Kit and QIAshredder (Qiagen). cDNA was produced in the pol region of nfl_HIV-1 and from the tag region flanking the 5′LTR in the pREC_cplt_R/U5/gag/Δpol vector using the following protocol: 5 μL of extracted RNA was added to 2 μL of antisense primer (20 pmol/μL) and cycled for 88°C for 2 min, 70°C for 10 min, 55°C for 10 min, 37°C for 10 min, and 4°C hold. Next, 5× first strand buffer (Invitrogen), 0.1 M dTT (Invitrogen), and 10 mM dNTPs were added to each reaction and cycled at 25°C for 10 min, 42°C for 2 min, and a 4°C hold. Finally, MMLV RT (Invitrogen) was added to the reaction and cycled at 42°C for 1 h, 70°C for 15 min, and a 4°C hold. Beta-globin was also reverse-transcribed from cellular RNA using random primers as a control [18].

Figure 6: Detection of HIV-1 production in VSV-G env pathway where the complementary viral subgenomes were separated in cytoplasm and capsid. (A) Three different coexisting patterns of HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs: cplt_R/U5/gag/Δpol sgRNA produced in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_ cplt_R/U5/gag/Δpol vector, and nfl sgRNA located in viral core through VSV-G/HIV-1 envelope-mediated entry (Condition V); nfl sgRNA produced in cytoplasm of U87.CD4.CXCR4 cells through transfection of pREC_nfl_NL4-3 vector, and cplt_R/U5/gag/Δpol sgRNA located in viral core through VSV-G envelope-mediated entry (Condition VI); and both sgRNAs are in same viral core wrapped with HIV envelopes as a positive control. (B) Real-time RT-PCR detection of sgRNAs in U87.CD4.CXCR4 cells infected/transfected in different combinations. (C) RT assay detection of HIV-1 virus production post-infection/transfection of U87.CD4.CXCR4 cells.

 

Taqman real-time PCR was performed on the cDNA that was described in the paragraph above. Briefly, 5 μL of a 1:5 dilution of cDNA was added to 1× Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the appropriate primers (300 nM/reaction) and probes (100 nM/reaction) to specif¬ically detect either the pol or tag region. Probes were labeled with FAM/MGBNFQ (Applied Biosystems) and were designed in part using Primer Express software (Applied Biosystems) [18]. Samples were run on an ABI PRISM 7700 sequence detection system in a 96-well format (50°C 2 min, 95°C 10 min, and 40 cycles of 95°C for 15 s. and 58°C for 1 min) and analyzed using SDS 1.9.1 software (Applied Biosystems). Samples were quantitated based on cDNA standards run alongside the samples with known copy numbers based on RNA concentration. To create cDNA standards, a region from pNL4–3 pol and a region of pREC_ cplt_R/U5/gag/Δpol were cloned into the pCR2.1-TOPO vector (Invitrogen) and transcribed with T7 polymerase using a MEGAscript transcription kit (Ambion, Austin, TX, USA). RNA produced during transcription was quantitated and diluted by 10-fold serial dilutions to create standards prior to reverse transcription. RNA was reverse-transcribed using the same protocol to make cDNA which was then used as standards in real-time PCR alongside the samples. β-globin was also amplified by real-time PCR from cDNA of cell lysates as a way to standardize cDNA input into the real-time PCR reactions. 

Figure 7: HIV-1 virus production in dual infection with fused virions. (A) Virus particles containing the complementary HIV-1 viral sgRNAs (i.e. virus #1 and #3, virus #2 and #4) were fused through liposome, and then infected the target cells via either HIV-1 or VSV-G envelope pathway. (B) RT assay detection of HIV-1 virus production in U87.CD4.CXCR4 cells infected with fused viruses through HIV-1 or VSV-G envelope pathway. 

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Results 


Construction of virus particles harboring partial HIV-1 genomes
In order to investigate the role of HIV-1 viral core in the process of reverse transcription, we devised a methodology to produce several virus particles harboring different HIV-1 subgenomes that require trans-complementation between these viral sgRNAs to complete reverse transcription and generate productive virus infections. As described in Figure 2, virus #1 was produced from 293T cell co-transfected with the pREC_cplt_R/U5/gag/Δpol to produce and package the cplt_R/U5/gag/Δpol complementing sgRNA, the pΔR8.91_gag/pol to produce the core proteins and viral enzymes, and pREC_HIV_env to pseudotype these virus particles with HIV-1 Envelope glycoproteins. pREC_cplt_R/U5/gag/Δpol is a new version of cplt vector containing HIV-1 R, U5, gag and partial pol sequences, which has higher packaging efficiency [22]. Virus #2 was the same as virus #1 in that it only encapsidated the cplt_R/U5/gag/Δpol RNA but was psuedotyped with VSV-G (from pMD.G) rather than with HIV-1 Env. Virus #3 was produced from 293T cells transfected with pREC_nfl_ NL4-3 to generate a nfl sgRNA for encapsidation as well as produce the entire HIV-1 proteome for proper virus particle assembly. 

The endocytic pathway through clathrin-mediated endocytosis is a basic mechanism by which the virus core is delivered into the target cell; this pathway is employed by many kinds of viruses including influenza viruses, adenoviruses, parvoviruses, and the Sindbis virus, etc. [23,24]. Incorporation of VSV-G allows viruses to enter the target cell using the endocytic low pH pathway. RT defects of HIV-1 caused by mutations in the capsid can be efficiently restored when pseudotyped with the VSV-G envelope glycoprotein [25], suggesting that the VSV-G pathway might provide some kind of protection of the viral genome from degradation by host enzymes before/during reverse transcription. Based on these studies, we generated two additional viruses pseudotyped with VSV-G. Virus #4 was the same as virus #3 but additionally pseudotyped with VSV-G. Finally, virus #5 served as a positive control and harbored both the HIV-1 nfl and cplt_R/U5/gag/Δpol sgRNAs. Since the viral RNAs differ in sequence, we could perform RT-real-time PCR to detect the sgRNA copy numbers in viruses by using probes specific for the two sgRNAs [18]. As showed in Figure 3A, viruss #1 and #2 only harbored the cplt_R/U5/gag/Δpol sgRNA, viruses #3 and #4 had only nfl sgRNA, virus #5 was positive for both sgRNAs, and the two sgRNAs were packaged into virus particles at similar efficiencies (i.e. 109-10 copies per ml culture).

A virtual TCID50 assay was performed on all virus particles using a methodology previously described [21]. Briefly, the serial dilutions of virus stocks followed by RT activity assay had a highly significant direct correlation of viral RT value to the infectious titer (TCID50 value) of same virus stocks. RT activity can served as a surrogate for viral RNA copies or p24 antigen content for over 50 primary HIV-1 isolates. Since virus #5 is infectious but harbors the same virus components of virus #1-4, we compare the serial dilutions of RT activity of the latter with the infections titer of virus #5 (Figure 3B), which was used to normalize the amount of virus input for the infection. 

There is no virus production following dual infection with the two complementary virus particles
In a previous study [18], we demonstrated that virus #5 (containing both HIV-1 cplt and nfl sgRNAs) could successfully complete reverse transcription and produce a wild type proviral DNA for integration and subsequent virus production. In this study, we utilized the same nfl sgRNA and an improved version of cplt sgRNA (cplt_R/U5/gag/Δpol) with higher packaging efficiency to investigate whether the two complementary HIV-1 genomes packaged into separate virus particles could still complement each other for reverse transcription following dual infection of susceptible U87.CD4.CXCR4 cells. The two virus particles (virus #1 and #3 [Condition I], or virus #2 and #4 [Condition II]) were normalized based on their virtual TCID50, and co-infected U87.CD4.CXCR4 cell culture with 10 MOI (Figure 4A) (Please note that the higher MOI caused significant rapid cell death). The post-infection cultures were monitored over 14 days by RT activity in the supernatant. We performed RT-real-time PCR and quantify both the cplt_R/U5/gag/Δpol and nfl sgRNAs in the cytoplasm of U87.CD4.CXCR4 cells at 48 hours post-infection/transfection. The results showed both of sgRNAs successfully entered into the susceptible cells with similar amount of the two sgRNA species in condition I, II, and the positive control (i.e. monoinfection with virus #5, containing both nfl and cplt_R/U5/gag/Δpol sgRNAs within one viral core) (Figure 4B), however, neither the HIV envelope glycoprotein nor VSV-G pseudotyped-virus co-infection of U87.CD4.CXCR4 cells established viral infection in two weeks’ period of culture, while the infection with virus #5 produced infectious viruses shortly after the inoculation (Figure 4C). We have previously shown that the cplt RNA, a previous version of cplt_R/U5/gag/Δpol can initiate reverse transcription and act as a template for (-) strand strong stop DNA synthesis [18]. Here, we again verified that reverse transcription could be initiated in both virus #1 and virus #2 by detecting (-) strong stop DNA product at 12 h post infection (data not shown).

Free sgRNA in the cytoplasm could not help the complementary sgRNA in the HIV-1 core to complete reverse transcription via either HIV-1 envelope glycoproteins or endocytic, VSV-G mediated pathway
Considering that the above non-infection of the culture was possibly due to the low chance of co-infection of two different viruses with 10 MOI in the same cells (please note the lower MOI did not produce infectious virus, either. Data not shown), the plasmids were transfected into U87.CD4.CXCR4 cells to generate either cplt_R/U5/gag/Δpol or nfl sgRNA in the cytoplasm, followed by the infection of the same cells with the viruses containing the corresponding complementary sgRNA. This strategy ensures plenty of at least one of the sgRNAs in the cytoplasm that will significantly increase chance to meet another sgRNA in the partially dismantled viral core or in the cytoplasm. 

Through different DNA transfection and virus exposures of U87.CD4.CXCR4 cells, we provided the HIV-1 nfl and cplt sgRNAs in two contexts: (A) cplt_R/U5/gag/Δpol sgRNA produced from transfected pREC_cplt_R/U5/gag/Δpol plasmid and nfl RNA derived from infection with virus #3 (Condition III), and (B) nfl RNA from transfected pREC_nfl_ NL4-3 plasmid and cplt_R/U5/gag/Δpol RNA from virus #1 (Condition IV) (Figure 5A). By using real-time PCR, in condition III, we detected 104-5/ml of nfl sgRNA and significant higher level of cplt_R/U5/gag/Δpol sgRNA (~109/ml) in the cell culture. Similarly, in condition IV, significant higher level of nfl sgRNA were also detected. The post-infection/transfection cultures were again monitored over 14 days by RT activity in the supernatant. However, there was no infectious virus produced from either cultures (Figure 5C). These findings suggest that, in HIV envelope entry pathway, the free subgenomic RNA, either cplt_R/U5/gag/Δpol or nfl, in the cytoplasm could not help the complementary sgRNA in the HIV-1 core to complete the reverse transcription process.

We then used VSV-G pseudotyped viruses (i.e. virus #4, containing nfl sgRNA, or #2, containing cplt_R/U5/gag/Δpol sgRNA) to infect the U87.CD4.CXCR4 cells which expressed cplt_R/U5/gag/Δpol or nfl sgRNA through transfection of the corresponding plasmids (Condition V or VI). RT-real time PCR results again indicated that the two complementary viral genomes, cplt_R/U5/gag/Δpol and nfl sgRNAs, were found in U87.CD4.CXCR4 cells (Figure 6B). However, this strategy did not produce infectious virus, either (Figure 6C). This result suggested that the endocytic pathway for entry again did not appear to permit the free sgRNA in the cytoplasm to the complementary sgRNA in the viral core to complete the reverse transcription process.

Complementation of reverse transcription via fusion of VSV-G pseudotyped virus particle prior to virus entry
In a previous study, we discovered that co-packaging of the cplt and nfl RNA subgenomes into the same virus particle resulted in complementation of the reverse transcription process and production of fully infectious, wild type virus and thus showed that they could help one another produce infectious viruses. However, in the present study, the two complementary viral genomes, nfl and cplt_R/U5/gag/Δpol RNA, were wrapped into separate capsids within separate virus particles. As described above, the failure of establishment of infection through dual infection is possibly due to the low chance for the two different viral cores to meet each other in the cytoplasm. However, the transfection/infection strategy should have provided plenty of chance for the two sgRNA to meet each other if the transfection-produced sgRNA could penetrate the partially dismantled viral core, or the sgRNA in the virus particle released from the viral core (note the cplt sgRNA containing virus could initiate the reverse transcription, resulting in the breakdown of its viral core) which should result in productive infection if the reverse transcription could occur in the cytoplasm. Therefore, it suggested that the viral core is indeed important for the HIV-1 reverse transcription process and the reverse transcription could not occur in the cytoplasm. 

To further investigate the possibility of viral core fusion during the reverse transcription process, we mixed the two viral cores through inoculating virus #1 with #3 (condition VII, HIV envelope entry pathway) or virus #2 with #4 (condition VIII, VSV-G envelope entry pathway) in the presence of liposome overnight and then infected U87.CD4.CXCR4 cells (Figure 7A). This procedure likely generates large enveloped virus-like particles with a membrane surrounding two or more viral cores. If these cores mix and form a new functional viral core, complementation of reverse transcription could occur with de novo infection and result in production of replication competent virus. Surprisingly, when the VSV-G pseudotyped particles (condition VIII) were fused with liposome, RT activity was detected in the supernatant by day 10 and peaked at day 14 post-infection (Figure 7B). This virus was successfully subsequently passaged on new U87.CD4.CXCR4 cells. However, the fused HIV enveloped virus (condition VII) didn’t show productive virus infection. The core mixing should have occurred in both conditions VII and VIII considering that liposome treatment is unlikely to favor more fusion in one condition over the other. Within the fused virus particles, it is unlikely that the virus cores from either condition would have different properties. Because the reverse transcription could not occur in the cytoplasm after viral core breakdown, these results suggest that, in the HIV envelope pathway, before and after the multiple core containing virus entry into the host cell, the viral cores did not fuse to form new functional viral cores, instead, they broke down, resulting in the degradation of sgRNAs by hostile cellular enzymes in the host cytoplasm. In contrast, in the VSV-G pathway, the virus particles were endocytosed through VSV-G envelope glycoproteins to form early endosomes where the sgRNAs were released from the viral cores and accomplished the reverse transcription process in an unknown mechanism (Figure 7A). 

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Discussion 


The events that take place after entry of the HIV-1 viral core are somewhat unclear. It was once held that the core uncoated immediately post-fusion and release into the cytoplasm [12-14]. However, some studies showed that TRIM5? can bind to polymeric CA, resulting in premature core disassembly and mediation of the RTC to proteosomal degradation, thus blocking reverse transcription [26]. It has also been shown that mutations that reduce the stability of the viral core are deleterious for viral infectivity and reverse transcription [27-31]. Other studies showed that the incoming capsid seems to retain an intact structure during its journey from the cell surface to the nucleus, reverse transcription happened within an integral capsid shell, and uncoating occurs at the nuclear pore upon completion of RT [16,25,15,32]. Thus, viral DNA synthesis and routing of HIV-1 RTCs/PICs to the nuclear membrane might be two independent events. Uncoating of the HIV-1 core is possibly the last step before the PICs across the nuclear pore, taking place after the DNA Flap formation that promotes it [8, 12,32,33]. This implies that the HIV-1 viral capsid might also play an important role in transporting the PICs into the nucleus. 

In the present study, we modified our previously constructed unique system [18] to specifically explore the function of the viral capsid during HIV-1 reverse transcription, whether the reverse transcription can occur in the host cytoplasm, and whether the different entry pathway will cause different consequence post-viral entry. The HIV-1 NL4-3 genome was split into two parts, nfl and cplt_R/U5/gag/Δpol sgRNAs, that are complementary to each other and can accomplish reverse transcription via strand transfer. Various virus particles with functional viral capsids were created to contain the two sgRNAs and wrapped with HIV-1 or VSV-G envelopes. We then set up several different coexisting patterns for the two sgRNAs in the susceptible cells through dual infection in the presence or absence of liposome, or infection/transfection (i.e. one sgRNA in the viral core, and the other sgRNA in the cytoplasm, or both in the viral core together or separately). The advantage of this system is that the two complementary HIV-1 sgRNAs can be separated in their specific locations; we can thereby distinguish whether reverse transcription occurs in the viral core, in the cytoplasm, or in the fused viral core. 

If reverse transcription occurs in the cytoplasm, the RNA genome (either cplt_R/U5/gag/Δpol or nfl) within the viral core should be released, meet its complementary genome in the cytoplasm. The two genomes should then cooperate to complete the process of reverse transcription. However, none of our coexisting patterns with the HIV-1 sgRNAs located in the cytoplasm (through transfection) showed production of infectious virus, suggesting that there was no successful completion of reverse transcription in the cytoplasm. 

On the other hand, the viral cores might fuse, resulting in meeting of the two sgRNAs and completion of reverse transcription within the fused viral core. However, our results showed that the co-infection with two sgRNA-containing viruses didn’t produce any infectious virus, even the fused virions resulting in two sgRNA-containing viral cores confined in one virus particle did not produce infectious virus, either. Thus, the viral cores do not appear to be capable of fusing to complete the first-strand transfer during reverse transcription. Among all of the coexisting patterns, only two sgRNAs located in the same capsid can produce replication competent viruses through the HIV-1 envelope pathway. This serves as confirmation that structural integrity of viral core is necessary for successful viral reverse transcription in HIV-1 replication. The study on the function of the HIV core suggested that the isolated core is active for reverse transcription [34]. In this study, since the cplt_R/U5/gag/Δpol RNA-containing virions (virus #1 and #2) contain sgRNAs from R to the middle of the pol region, including the primer binding site (PBS), and all of the viral proteins, reverse transcription was initiated but not completed. It will be interesting to investigate whether these viral cores are uncoated at this point in the process.

Pathways that the HIV-1 viral capsid may follow post-fusion or endocytosis into the host cell are not clearly defined. There is evidence that the cellular cytoskeleton facilitates early steps of HIV-1 infection [4], and that the capsid is one of the main targets of regulation. The viral cores are deposited in the cell cytoplasm after entry via fusion through the HIV-1 envelope. The capsid cores of HIV-1 and similar retroviruses bind dynein motors and traffic along microtubules towards the nucleus. On the contrary, HIV-1 virions carrying VSV-G are predicted to enter cells through an endocytic pathway, since this is the normal route of VSV infection. These chimeric viruses are capable of infecting a wide variety of cell types and were recently used to enhance the utility of HIV-1-based gene delivery vectors [35]. By tracking fluorescent viral complexes in living cells infected with the VSV-G pseudotyped virus, researchers demonstrated that short distance and rapid movements of HIV-1 cores are characteristic for an actin-polymerization-dependent transport [19,32]. The microtubule network also supports long distance movement of HIV-1 core complexes. However, actin-cable-dependent trafficking systems recruited by HIV-1 complexes that are delivered through HIV Env and VSV-G may differ. Indeed, HIV-1 infection is blocked at the RT level in cells that express siRNAs to the Arp2/3 actin nucleator complex, which inhibits polymerization of actin. This block is no longer observed when using VSV-G pseudotyped HIV-1 [36]. 

Viruses entering target cells through the VSV-G pathway will first form endosomes and then lysosomes (which contain enzymes), and virions will be limited within a closed subcellular structure. Brun S. et al.[25] found that HIV-1 viruses bearing mutated capsids that disrupt virus infectivity via impairment of core assembly and stability could be efficiently restored by pseudotyping with the VSV-G envelope glycoprotein through the endocytic pathway instead of the HIV-1 Env-mediated fusion process [25]. This indicates that the pathway that viral core shunting in the target cell may influence subsequent function of the viral core, and possibly protect HIV-1 RNA from degradation by host enzymes. Our present study found that, even though co-infections with the two different VSV-G-viruses didn’t produce any infectious virus, infectious progeny was successfully created when the two virions were fused by liposomes before infection, which ensured the two cores entered the host cell together. This result indicates that defects in reverse transcription can be restored using the VSV-G mediated endocytic pathway, but the two capsids must be in close proximity in order for this to happen. Because it has been demonstrated that the reverse transcription cannot occur in the cytoplasm and the viral cores cannot fuse to form new functional viral cores, it is highly likely that either the two viral cores/capsids are broken down in the closed subcellular structure (i.e. endosome/lysosome), or that the uncoating is triggered by reverse transcription, resulting in the release of the two complementary HIV-1 RNA subgenomes and completion of the first-strand transfer. On the other hand, the fused virus bearing HIV-1 envelopes may be set apart post-entry and degraded by the host enzymes in the cytoplasm, resulting in non-productive infection. Therefore, an intact state of the viral core is important for viral reverse transcription in natural HIV-1 infection, and uncoating might only entail the opening of viral core, rather than complete degradation of the capsid, so that the necessary materials can access the reverse transcription machinery.

Overall, HIV-1 reverse transcription is a complex and highly ordered process. HIV-1 RNA is vulnerable and easily broken when exposed to the numerous enzymes in the cytoplasm; its viral core is thus an ideal site to protect the viral RNA and confine the necessary materials within a limited space in order to ensure successful reverse transcription. The number of reverse transcriptase per capsid (80 to 120) in the HIV-1 reverse transcription complex (RTC) makes this especially true. Considering the need to maintain stoichiometry in the reactions between enzymes and viral templates for rate limiting steps such as polymerization pauses, strand transfers, and formation of the central DNA Flap, it is only logical that reverse transcription occurs within an integral viral core/capsid structure in the cytoplasm of host cells [37-39]. Even if the VSV-G pathway helps the capsids that contain two complementary genomes complete reverse transcription via strand transfer, a relatively enclosed space (i.e. lysosome) is necessary for the first-strand transfer. The role of CA in PIC production and transport towards the nucleus still needs elucidation. 

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Acknowledgements 


This study was supported by research grants awarded to Y.G. and E.J.A. (NIH/NIAID AI84816 and AI49170), and to W.H (Y201023, Department of Public Health of Jiangsu Province, China).

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