Simulation of a HIV-1 virus infection of a CD4+T lymphocyte by Monte Carlo

ABSTRACT

The Human Inmunodeficiency Virus (HIV) is a retrovirus which preferentially infects CD4+T lymphocytes of the human inmune system during its replication cycle. Although the cycle is similar to other retroviruses, there are still aspects which remain unknown, so further research is still needed. Computerised biology brings the possibility to reproduce with low resources the virus replication cycle, focusing on those steps in which real laboratory conditions are difficult to achieve. The software HIV.pro has been developed to simulate by Monte Carlo methods the replication cycle of HIV-1 virus, providing estimators of HIV protein concentrations in the nucleous and cytoplasm, spliced and unspliced mrna concentrations, viral burst size time and number of transcriptions for first virus release. Simulations have estimated that maximum transcription rate of 185.2 kb/min is achieved with a threshold level of protein [tat]=200 nM in the nucleoplasm and completly splicing is avoided with a threshold level of protein [rev]=2 nM in the nucleoplasm. Viral burst size has been estimated as 6378 ± 65 virions per infected cell, with an average time per 1st virus release of 32 h (3 σ), comparable with other published studies. Total number of transcribed mrnas presents differences, with only 1 mrnas (1kb) and 77 mrnas (1.8kb) compared with around 42000 mrnas (4kb) and 30000 mrnas (9kb), showing that 1kb mrna could be an initiator of the replication cycle or a residual mrna which translate occasionally nef protein. Furthermore, the core process of virus replication activity is based on 4kb and 9kb, where structural proteins are mainly translated. Virus progenie present variable levels of [vif] and [vpr] proteins, depending on the protein levels in the cytoplasm during budding stage. A 8 % of virus produced has indeed no concentration of vif protein, while a 34% has concentrations of vpr protein lower than those previously reported. HIV-1.pro software simulates acuratelly the intracelullar process of virus infection, providing reliable results of virus infectivity.

K eywords HIV-1 virology · virus cycle · Monte Carlo

1 Introduction

Human Inmunodeficiency Virus (HIV) is a retrovirus who needs a host-cell, such as CD4+T lymphocytes of the inmune system, for protein synthesis and virus assembly during its replication cycle. HIV stays in a latent state in the host body during several years and without effective treatment, reduces the level of CD4+T lymphocytes, colapsing the inmune system, therefore several oportunistic diseases appear, what it is known asthe AIDS (Acquired ImmunoDeficiency Syndrome) [1, 2]. Since being identified almost three decades ago, the HIV-1 pandemic, also called the main (M) group has infected at least 60 million people and caused more than 25 million deaths [3]. Although antiretroviral treatment (ART) reduces considerably spread of HIV and death by AIDS, effective vaccines and curative treatments are still uncertain, being AIDS a worldwide health threat [4, 5]

HIV produces more than 40 different polycistronic mrnas during RNA polymerase II transcription stage encoding all the proteins that the virus needs for replication, such as regulatory enzymes (nef, rev, tat, vpr, vif) or structural proteins (vpu/env and gagpol) [6]. HIV genome has been studied previously reporting these proteins encoded in different reading frames, including ribosomal gag-pol frameshift stem-loops or vpu/env ribosomal sp-stem loops, during protein synthesis [7]

Computerised biology brings the possibility to reproduce with low resources the virus replication cycle, focusing on those steps in which real laboratory conditions are difficult to achieve. Simulations have been applied in the past to model viral spread, CD4+T cell concentrations and protein levels in human cells and tissues [8, 9, 10, 11] Other studies have studied in a multi-scale modeling HIV infection and effects of anti-retroviral therapies (ART) in intracellular and extracellular models [12, 13]

Monte Carlo methods are based on an approximation of the final solution of a system through random numbers, in which the processes can be described through events with known probabilities. These methods have been extensively applied to virus spread models [14, 15]. Although Monte Carlo methods have been mainly used in epidemiological studies [16], modelling virus infection at cell dimension can provide accurate estimators of cell parameters and virus infectivity [17, 18].

In this report, the software HIV.pro, a fortran 90 / C++ code, is described, focusing on the results of the Monte Carlo simulations: viral burst size, time for first virus release and protein concentrations during a period of 72h after infection.

2 HIV-1 virus

2.1 Introduction

HIV (Human Inmunodeficiency Virus) is a virion of the family Retroviridiae Lentivirus, with two main types (HIV-1 and HIV-2) and multiple groups and subgroups within them. HIV-1, group M, is the most common and subject of most studies [19].

HIV-1 genome has 9719 nucleotides, encoding several genes in 3 reading frames, as observed in figure 1. These genes decode structural proteins (gag/env), enzymes (pol), transcription promoters (tat), export signals (rev) and other regulatory proteins (nef/vif/vpr/vpu)[20]. Table 1 describes the function of these proteins.

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Figure 1: Scheme of the HIV-1 genes and position in the reading frame

HIV is composed of a spherical lipid membrane, a matrix protein (MA), a conical capsid (CA) and a nucleocapside (NC), which protects a double single stranded RNA genome (vRNA). On the external surface of the lipid membrane, there are placed glycoprotein trimer spikes, used by the virus for fusion into cells. Inside the nucleocapsid, there are several enzymes such as protease (PR), reverse-transcriptase (RT) and integrase (IN), as other virus proteins (nef/vif/vpr) in different concentrations [21].

2.2 HIV-1 virus cycle

HIV virus attaches to the CD4 co-receptors (CCR5 or CXCR4) and fuses with the cell membrane infecting the cell. After this, the viral RNA is reverse-transcripted into cDNA, forming a pre-integration complex (PIC) which is integrated randomly into the DNA of the host-cell, being in a latent state until transcription is initiated. Several transcription factors attach to the LTR promoter, starting to recruit RNAPII polymerases, transcribing continuously pre-mrnas. These pre-mrnas are spliced, capped and polyadenilated and then exported to the cytoplasm for protein synthesis. Some of these proteins are assembled into new viruses, while other proteins acts as virus enzymes, as tat protein, which is a transcription activator or rev protein which export unspliced or partially spliced mrnas to the cytoplasm (figure 2).

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Table 1: Summary of the main genes and protein function of HIV-1[22, 23, 24]

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Figure 2: HIV-1 replication cycle in a CD4 T-lymphocyte: (1) Attachment to the CD4 co-receptors (CCR5 /CXCR4), (2) fusion and uncoating of vRNA, enzymes and proteins, (3) reverse transcription of RNA and pre-integration complex (PIC) assembly, (4) integration of PIC into host-cell DNA, (5) transcription initiation, (6) splicing of pre-mrnas, (7) export of mrna to cytoplasm, (8) translation of mrnas and protein synthesis, (9) import of [rev/tat] proteins into the nucleoplasm, (10) assembly of HIV viruses, vRNA packaging and budding from the host cell and (11) virus maturation - disassembling by protease enzyme and re-assembling (Image produced with https://app.biorender.com

HIV-1 assembly and RNA packaging are complex processes in the virus cycle still unknown. The HIV-1 vRNA has a packaging signal (Ψ), that it is requiered under given conditions for gag/gagpol recruiting during virus assembly [27, 28]. Packaging iniziation complex (PIC) is formed when two vRNA dimerize preferentially through non-covalently intermolecular base-pairing of the dimerization initiation sequence (DIS) [29]. These PICs molecules starts recruiting gag and gag-pol glycoproteins which assemble preferentially with Ψ-containing RNAs, multimerizing through protein- protein interactions and then at the plasma mebrane of host-cells by myristoylation at their N-termini of gag and gagpol proteins [30, 31, 32], as observed in fie3. Some studies have proved that altho1Wl DIS is a central element for dimerization, in abscence of DIS, dimerization can still occur, but with reduced kinetics and unidentified mechanisms [33]

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R&ure3: HIV-1 virus protein assembly in the cell membrane: (1) env trimer,composed by &lycoproteins gp120 an:! &P41 (transmembrane re&ion), (2) reyristate anchorm& on cell membrane, (3) &a& and&a&pol polypeptides and (4) vRNA dirner (lma&e produced with https:llapp.bioren:ler.com)

3 Software HIV.pro

3.1 Introduction

HIV.pro software is a fortran.90/C++ code which simulates virus cycle replications in a CD4-T lymphocyte. The C++ block is focused in the analysis of the NCBI genome, the identification of the HIV proteins and secondary structures. The fortran 90 block simulates by Monte Carlo the virus replication cycle in an intracellular scale: infection, transcription, splicing, mrna export to the cytoplasm, protein translation, tat/rev transport, RNA packaging and assembly of new inmature virions (see fig. 9) [24]

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Figure 4: HIV.pro software modules in fortran90/C++.

The software has been used to simulate by Monte Carlo an infection by a single HIV-1 virus in a CD4+T lymphocyte during all the life-time of the cell of 72h, estimating HIV protein concentrations, viral burst size and number of spliced and unspliced mrnas in the cell.

3.2 Module Transcription

RNAPII polymerases produce continuously after infection pre-mrnas with a full-length of 9kb, which can be exported to the cytoplasm or accumulated in the nucleous undergoing splicing by the spliceosome complex and splicing factors. Transcription rate is normally distributed between [0.3-4] kb/min. In the case of a first infection, basal transcription rate is ω 0 =1.9 kb/min, with a transcription time of 5.12 min per premrna for the HIV-1 genome (9719 bp) [34]. It has been assumed that, as the provirus genome is composed by two sequences, the density of RNAPII polymerases transcribing them at the same time is 2, as presented in figure 5.

The HIV transactivating protein Tat regulates transcription, increasing the elongation rate and consequently the number of premrnas in the nucleous. Tat protein binds to the TAR element in the 5’LTR region of the genome. After tat protein is translated, they are imported to the nucleous, as other regulatory protein, binding to TAR elements of the nascent premrna and increasing the rate of transcription [36].

Tat protein is intron-exon dependent, formed with a fragment of exon 4 and another from exon 7, including in some cases exon 6. Tat protein presents therefore several forms, which can be reduced at least into tat1/tat2. Tat1 is a 86aa protein formed from exon 4 and exon 7b in 1.8kb mrnas; tat2 is a 69aa protein formed exclusively from exon 4E in 4kb mrnas [23, 37, 38, 24]. It has been assumed that both proteins activate equally TAR element.

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