Subnormal osmolarity may underlie microcephaly in Zika viral infections

Qiuyun Liu Man Tang Yanchao Zhou Yuchuan Wang Xiaoxia Li

【Abstract】Leucines accounted for 9.144% of the Zika viral full length protein with over 300 leucines. The amino acid residue lacks γ methyl group， thus devoid of the σ-σ hyperconjugation in beta carbon， as well as strong van der Waals interactions with the carbonyl group of leucine. This results in the low cation affinity with carbonyl oxygen， and un-enhanced secondary chemical bonding with the oxygen atom. The subnormal osmolarity generated in the presence of leucine rich proteins and degraded peptides would affect the development of the brain. A carbohydrate diet could be effective in the prevention of viral infections.

【Key words】Subnormal osmolarity； Microcephaly； Leucine

Zika viral infections were suspected to be linked to microcephaly and other brain defects in babies. In the polyprotein of Zika virus with a primary sequence of 3423 amino acids （GenBank accession no. KJ776791）， basic and acidic amino acids were of similar amount， while leucines accounted for 9.144% of the full length protein. The microcephaly that can cause permanent brain damage in newborns inflicted with Zika virus might be associated with the lack of γ methyl group and hyperconjugation in beta carbon， and consequently un-enhanced cation affinity of the carbonyl group in leucines， resulting in subnormal osmotic pressure.

While leucines in most proteins are harmless and help to form higher-order structures， they can form potent antimicrobial peptides. Leucine has delta methyl groups， and there is hyperconjugation in the distal end of the side group， which enables intermolecular interactions together with hydrophobic forces. This hypothesis is corroborated by bond lengths （Gould R.O. et al.， 1985）， NMR chemical shifts and reports of leucine zipper motifs facilitating dimerization.

Essential amino acids are defined based on in vivo biosynthesis， which include leucine， isoleucine， valine， lysine， phenylalanine， threonine， methionine， tryptophan and histidine in humans. Although they are all functionally important， they clearly fulfill some roles through the lack of synthesis in humans and animals from an evolutionary point of view. They are well conserved over tens of millions of years. The mortality rates of 1918 Spanish flu in China were reported to be much lower than in western countries， which may be related to the plant-based and starch rich diet in pre-industrial China as pre-exposure to the virus was not reported. Americans lifespan was increased by several years in in the difficult times of 1930 to 1933 when people experienced the Great Depression and hunger. Animal and model organism studies also pointed to a vital role of nutrition on lifespan. Essential amino acids could be one of the key factors in this regard. It may well be the consequences of selection in the animal kingdom for antiviral purposes. For instance， the multiple basic amino acid sequence of the hemagglutinin cleavage site （RERRRKKR↓GLF） is associated with the high virulence of the H5 avian influenza virus in animals. Most proteins are negatively charged， and do not have high content of basic amino acids.

Leucine may have no strong cation binding tendency including protons， and its solubility is poor. It is rich in many proteins. However proteins rich in Leu and basic amino acids may be very toxic to the cells， and this toxicity may be attenuated by acidic amino acids， etc. The leucine content is around 15% in the virulence protein VP24 of Ebola virus. The isoleucine content is around 16% in the virulence peptide orf6 of SARS virus. The GP5 protein in Rabies virus has overrepresented LK and VK di-amino acids.

The σ-σ hyperconjugation in valine， and interactions between γ methyl group and carbonyl group were strongly supported by previous studies including experimental data such as NMR chemical shifts， coupling constant and bond lengths （Suardíaz R. et al.， 2007）. The carbonyl carbon of isoleucine was one of the most shielded among the amino acids tested as shown by the chemical shift anisotropy of σ22 element of the tensor， which supports the presence of stronger hydrogen bonds with the carbonyl oxygen in isoleucine than in leucine （Gu Z. et al.， 2002）. Valine has two γ methyl groups， and the strong hyperconjugation may render the carbonyl group to be strongly cation binding， which may include divalent cations. The increasingly longer C═O bond lengths in Leu， Ile and Val supported the formation of stronger secondary bonds with carbonyl oxygen in Val and Ile （Gu Z. et al.， 2002）. The strength and bond lengths of secondary bonds are between covalent bonds and van der Waals forces， and secondary bonds are prevalent in the natural world.

In the case of West Nile virus， treatments to pharmacologically stimulate autophagy were effective in reducing the neurotoxic effects in individuals， suggesting that viral proteins and peptides may be the culprit for viral virulence， instead of cytokine storm alone.

The hydrogen bonding potentials of various amino acids were previously documented （Gu Z. et al.， 2002）. The van der Waals forces between side groups and peptide groups were also studied. Trp rich antimicrobial peptides had broad and potent antimicrobial activity. The carbonyl oxygen of Thr may possess strong hydrogen bond capacity besides the hydroxyl group， and Thr possesses a γ methyl group. Amino acids such as phenylalanine and tyrosine possess conjugated π system with affinities to cations and photons. Histamine， an allergic substance at some circumstances， is derived from histidine by decarboxylation. Secondary chemical bonding may underlie the virulence of some viral proteins and peptides， which may generate abnormal osmotic pressure and disrupt cell homeostasis. Essential amino acids such as lysine and hydrophobic amino acids frequently feature in antimicrobial peptides， and are sometimes cytocidal to mammalian cells.

The microcephaly in newborns affected by Zika viral infections might be caused by the subnormal osmotic pressure， generated by the leucine abundant protein due to the lack of γ methyl group and hyperconjugation in beta carbon， and consequently un-enhanced cation affinity of the carbonyl group in leucine residues. The emergence of essential amino acids may be an evolutionary adaptation to cellular stresses and infectious diseases as peptides rich in essential amino acids are cytocidal in many previous reports， and carbohydrate only diet or carbohydrate rich and certain amino acid deficient diet could be effective prophylactic or early treatment regimens against Zika viral infections as it does not give rise to essential amino acids. Trials with other viral infections can also be conducted before or in the early phases of viral infections.

References：

[1]Gould R.O.，Gray A.M.，Taylor P.，Walkinshaw M.D.1989.Crystal environments and geometries of leucine，isoleucine，valine and phenylalanine provide estimates of minimum nonbonded contact and preferred van der Waals interaction distances.J Am Chem Soc，107（21）：5921-27.

[2]Suardíaz R.，Pérez C.，Vega J.M.，Fabián J.S.，Contreras R.H.2007.Theoretical Karplus Relat-ionships for vicinal coupling constants around χ1 in Valine.Chem Phys Lett，442：119–23.

[3]Gu Z.，Zambrano R.，Mcdermott A.2002.Hydrogen bonding of carboxyl groups in solid-state amino acids and peptides：comparison of carbon chemical shielding，infrared frequencies，and str-uctures.J Am Chem Soc，116（14）：6368-72.