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HIV-1 Drug Resistance Through Primordial Proteins of Nanobacteria By: Juan Montalvo Copyright May, 2013 The rate at which antibiotics such as prescription drugs and over the counter medication are being misused and overused. This contributes to microbial resistance and ultimately, virulence. Moreover, agricultural industries utilize growth producing antibiotics in poultry to stave off infection to presumably increase the overall life span and quality of their poultry. As the fourth largest poultry producer in the U.S., the Perdue company employs these practices.1 The effectiveness of important human drugs will be reduced as selective processes and mutation of microbial pathogens increase.

Alarmingly enough, patients infected with the human immunodeficiency virus (HIV) excrete highly resilient nanobacteria (NB) that are protected by a shell of primordial glycoproteins.2 Nanobacteria (NB) have been linked with patients having HIV. Studies have shown that the perineurium in HIV infected individuals is coated with apatite, a calcium phosphate shell that protects the NB.4 This is significant because metabolic proteases and signal transduction of a nerve are either interrupted or restricted. Given enough time, peripheral neuropathy may result, a condition in which nerves become damaged. Upon analysis of NB and biological surface interaction, it was found that cylindrical surfaces favor encapsulation by NB as opposed to extended plane surfaces.4 In other words, blood vessels and the perineurium serve as ideal surfaces with which to attach to. NB do so by self-assembling onto hydrophobic apatite to form a slime layer. As the permeability of the perineurium decreases, so too does its efficiency. Experiments were conducted to evaluate the effect of polarity on attachment.

Two human hairs and an optical fiber were positioned on mirror-polished titanium disks. They were all inoculated with a drop of nanospheres 60 nm in diameter. To ensure slow evaporation of the nanosuspension, the titanium disks were enclosed within Petri dishes. Slow evaporation increased contact time between the substrates and the nanospheres. This was meant to emulate the biological conditions that are likely to take place. Symmetric distribution of the nanospheres on the hydrophilic optical fiber occurred. The opposite was true of the hydrophobic human hairs.

Findings are have shown that because NB are present within Earth's upper atmosphere, it is correlated with reduced bone density in humans.2 With a large reservoir of HIV infected patients in sub-Saharan Africa, the spread of NB on a global scale has also been associated with an aggravation of symptoms in individuals with HIV, increasing virulence.4 The most common type of HIV is HIV-1. Essentially, the virus destroys the immune system of an infected individual by killing off the CD4 cells; which are involved in fending off disease. In particular, the HIV-1 virus bonds to CD4 cells and attaches itself to the outer membrane. It then injects RNA into the host CD4 cell. Within the cell its genetic material utilizes an enzyme called integrase to integrate the RNA into the host cell genome. The RNA template is then synthesized into DNA via reverse transcriptase and hijacks the host cells machinery to replicate itself until the CD4 host cell ruptures, enabling further infection of other CD4 cells. One way of treating HIV-1 is to inhibit integration into the host cell genome. Medications developed at targeting the integrase enzyme are integrase strand transfer inhibitors (INSTIs).3 Though INSTI's serve as an effective mode of treatment, HIV-1 is a so called retrovirus because it uses RNA as its genetic make-up and is the reason why finding a cure is very difficult and

elusive. RNA is highly unstable in an aqueous environment and does not have reliable mechanisms with which to repair flaws within the RNA sequence itself should it become damaged. As a result, the rate of mutation is very high and causes drugs to become less and less effective over time. Well-conserved amino acid residues such as Asp64, Asp116, and Glu152 are essential to the enzymatic structure of integrase.3 Based on these findings, this also serves a target to combat resistance. Another method of treating HIV-1 is to inhibit entry into the CD4 cell. Tyrosine sulfate intermediates place a cervical role of the virus into the cell, and as much, serves as a target. After the virus attaches itself to the CD4 cell, the HIV-1 gp120 glycoprotein binds to the CCR5 coreceptor that involves the interaction of two tyrosine sulfates.5 Compounds aimed at mimicking tyrosine sulfates contain a phenyl sulfonate-linker-aromatic motif that essentially make the target site sterically hindered. This makes HIV-1 binding of CD4 impossible. This experiment also helped revealed how HIV-1 evolved the ability to evade antibody recognition.5 With the ever increasing resistance of microbes towards antibiotics and growing evidence of a strong interplay of nanobacteria and HIV-1 virulence, the degree to which the entire world can be effected is very real and warrants further attention. Due to the high rate of mutation, HIV1 evades the bodies immunological response. An approach to treating HIV-1 is to target highly conserved regions of the retrovirus. In particular, targeting the structures responsible for viral binding serves as a viable means. The V3 loop of gp120 of HIV-1 is such a structure.6 It binds the virus to CCR5 or CXCR4 of CD4 cells. Upon NMR analysis, the V3 loop shows formation of an amphipathic turn comprised of hydrophobic and charge interactions. Studies have shown that bacterial lipopolysacchardies (LPS) inhibited binding of gp120.6

Alternatively, HIV-1 treatment of both wild-type and drug-resistant strains employ inhibition of protease dimerization. Alkyl tripeptides target the four-stranded antiparallel betasheet.7 A minimum length of the alkyl chain is required to inhibit dimerization which includes palmitoyl-Leu-Glu-Tyr.7 The HIV protease (HIV-PR) dimerization inhibitor functions by replacing the middle strand of the beta-sheet. Given that the HIV-PR is effective against HIV-1 mutants, it serves as a viable therapy.7 Also, protease inhibitors and reverse transcriptase inhibitors are used in HIV-1 treatment.

Lastly, a restriction factor such as APOBEC3G, inhibits HIV-1 replication. It accomplishes this by deaminating cytosine to uracil. HIV-1 reverse transcriptase then utilizes those uracils during reverse transcription to create G-A mutations on the viral genomic strand, resulting in hypermutations and deactivation of the virus.8 A protein, viral infectivity factor (Vif), disrupts APOBEC3G's deamination activity. Studies show Vif co-encapsidation with APOBEC3G produces sub-lethal effects on HIV-1 DNA.8 Several experiments were done: Model HIV-1 replication, deamination, and steady state rotational anisotropy assays.8

Though many treatments are available in combating HIV, it's genetic diversity makes it difficult to find a cure. The exact mechanism(s) by which this genetic diversity is being promoted is the subject of debate. One theory is that NB contribute to the genetic diversity in HIV, one that will be elaborated on later.9

The size of NB are miniscule (60-300 nm), and as such, identification of nucleic acid is difficult, leading many to question whether or not NB are alive. Three intrinsic characteristics of NB that are consistent with conventional biosystems and essentially conferring life are: 1. protection of a permeable apatite mineral shell, 2. protein based slime synthesis and growth in

response to environmental stress (similar to chemical variations in the blood), 3. sensitivity to visible light based on intensity and dose which is equivalent to biostimulatory standards. The latter characteristic has been shown to elucidate a response in mammalian cells which were observed to prevent the protein based slime synthesis of NB. Therapies aimed at reducing NB concentrations in the body could originate from the last two characteristics. Without the protein based slime, the bioadhesive capacity of NB decreases.9

Also, NB have the capability to harvest solar irradiation based on its chemical make-up. The proteins produced by NB are argued to be primordial proteins. Upon further analysis, the function of these primordial proteins were to collect and store nutrients as well as apatite components. Even more crucial, NB primordial proteins function in adhesion and surface sealing. This enables NB to survive in extreme environments. The allocation of calcium and phosphate minerals causes NB growth. This implies diffusion of the minerals across the protein based slime layer, thereby establishing a calcium-phosphate gradient to produce the mineral shell. The presence of NB within the blood stream may play a role in the interaction of the HIV virus and the host immune system. Calcium-phosphate has been shown to enhance the uptake of DNA and RNA in eukaryotic cells. Though poorly understood, the mechanism of uptake due to calcium-phosphate may be attributed to its interference of transport processes in the cytoplasm or across cellular and nuclear membranes. In 1973, calcium-phosphate was discovered to be a natural mediator of DNA in nucleic acid transfection.9

Without the collection, storage, and carrier function of primordial proteins, nucleic acid transfection of lymphocytes with viral nucleic acids would not be likely. Co-infection of NB and HIV can enhance virulence by increasing the rate at which lymphocytes are infected. In a figure

describing NB-HIV double infection, two NB (60-300 nm) readily diffuse through a human lymphocyte membrane and adhere to the nuclear membrane. Calcium-phosphate enriched NB facilitate the uptake of viral DNA by the nucleus. The protein based slime layer of the NB increase nuclear membrane fluidity and the surface area for points of contact with HIV. The longer the NB protein based slime layer resides on the nuclear membrane, the greater the chance of it creating a channel to facilitate passage of viral DNA.9

Apatite shell and slime envelopment of NB still requires further research designed at studying NB and HIV infected lymphocytes in depth. Future studies should also include the time it takes for HIV infection to occur in geographic zones with high and low concentrations of NB in the blood. So as to exclude coincident co-infection of HIV with increased NB concentrations, detection of NB with their slime envelope in HIV infected lymphocytes are required.9

High resolution imaging techniques that function in aqueous liquids such as environmental transmission electron microscopy (E-ETM) serve as a means with which to visualize nanoparticles. This can be used as a tool in clinical studies involving NB-HIV interactions. The central question in conducting such experiments is to know whether or not the possible presence of nucleic acids within the NB can travel though the apatite shell. Experiments have been conducted, suggesting that RNA from NB was released. More evidence is required and merits further research. The utilization of tracer liquids can determine the diameter of the pores located on the apatite shell.9

In Africa, the importance of NB in HIV infection also deserves attention. In particular, the use of human excretion in Sub-Saharan Africa (an area where HIV infection is high) agricultural irrigation, serves as a reservoir for viable NB. This is significant in that NB may

contribute to the increasing rate of HIV turnover due to transfection. Turnover of viral DNA by recombination events such as HIV is facilitated via NB which increases the chances of infection by two different strands of DNA. Also, NB are suspected of having an effect on the latency period of infected lymphocytes.9

Of the 30,000,000 HIV-infected Sub-Saharan Africans, NB excretion possesses a highly infective potential. This is significant in that re-incorporation of NB will likely increase HIV mortality by entry into lymphocytes. Given the possible epidemiological impact on such a massive scale, a global scrutiny of agricultural irrigation, fertilizers, and the environment for viable NB loads demands attention.9

References 1. Graham JP, Boland E, Silbergeld E. Growth promoting antibiotics in food animal production: an economic analysis. Public Health Reports 2007;79-87. 2. Sommer AP, Pavlåth† AE. Primordial proteins and HIV. Journal of Proteome Research 2005;633-636. 3. Xue W, Jin x, Ning L, et al. Exploring the molecular mechanism of cross-resistance to HIV-1 integrase strand transfer inhibitors by molecular dynamics simulation and residue interaction network analysis. Journal of Chemical Information and Modeling 2013;210-222. 4. Sommer A.P. Suffocation of Nerve Fibers by Living Nanovesicles: A Model Simulation, Journal of Biological Chemistry, (2004), 3: 3, 667-669. 5. Acharya P, Dogo-Isonagie C, and J.M. LaLonde Structure-Based Identification and Neutralization Mechanism of Tyrosine Sulfate Mimetics That Inhibit HIV-1 Entry, American Chemical Society, (2011), 6: 1069-1077. 6. Majerle A, Pristovsek P, Mancek-Keber M, and J. Roman Interaction of the HIV-1 gp120 Viral Protein V3 Loop with Bacterial Lipopolysaccharide, Journal of Biological Chemistry, (2011), 286: 29, 26228-26237. 7. Bannwarth L, Rose T, and Laure Dufau Dimer Disruption and Monomer Sequestration by Alkyl Tripeptides are Successful Strategies for Inhibiting Wild-Type and Multidrug-Resistant Mutated HIV-1 Proteases, American Chemical Society, (2009), 48: 379-387. 8. Feng Y, Love R.P., and Linda Chelicon HIV-1 Viral Infectivity Factor (Vif) Alters Processive Sinlge-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G, Journal of Biological Chemistry, (2012), 288: 9, 6083-6094. 9. Sommer AP. Primordial Proteins and HIV Part II. Journal of Proteome Research 2005; 10221024.

HIV-1 Drug Resistance Through Primordial Proteins of Nanobacteria