Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase.

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN's effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 µM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity.

d-bifunctional protein (d-BP) deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

Clinical effects of bovine lactoferrin on two canine cases with familial neutrophil dysfunction.

This study reported detailed clinical effects of bovine lactoferrin on 2 canine littermates (1 female and 1 male) with familial neutrophil dysfunction and an investigation of their genetic background. Clinical signs caused by severe upper respiratory bacterial infections were observed in these dogs. Oral administration of bovine lactoferrin for a long duration improved their clinical signs (severe uveitis in the female dog and coughing from pneumonia in the male dog). Their backcross dogs that have the same father didn't show clinical signs of bacterial infection. Neutrophil function tests revealed that the backcross dogs didn't have any disorders. It is likely that abnormal clinical signs are associated with neutrophil dysfunction in the colony, and the mother dog of these cases might be the genetic carrier of this dysfunction.

Oral administration of bovine lactoferrin upregulates neutrophil functions in a dog with familial ß2-integrin-related neutrophil dysfunction.

Lactoferrin, a glycoprotein present in neutrophils and exocrine secretions, plays important roles in host defense. Administration of bovine lactoferrin has been reported to modulate various neutrophil functions. We found a mixed-breed male dog with novel familial neutrophil dysfunction. The disorder was caused by a decrease of ß2-integrin expression encoding CD18 without mutation. Antibiotics therapy alone did not influence a series of neutrophil functions in the same dog. We examined the effects of oral administration of bovine lactoferrin on the neutrophil function and clinical symptoms in the same dog. Oral chronic administration of bovine lactoferrin increased neutrophilic ß2-integrin gene expression comparable to normal dogs, followed by the upregulation of surface CD18 expression. Concurrently, the superoxide production, phagocytic activity and adherence that were ß2-integrin-related neutrophil functions increased to normal canine levels. The chronic inflammation from bacterial upper respiratory infections and pneumonia was also alleviated in the dog. Our results indicate that oral treatment with bovine lactoferrin increases neutrophil ß2-integrin transcript level, leading to the upregulation of neutrophil functions and improvement of clinical symptoms in the dog with familial neutrophil dysfunction.

Canine neutrophil dysfunction caused by downregulation of beta2-integrin expression without mutation.

Canine leukocyte adhesion deficiency (CLAD) in Irish setters is caused by genetic defects of leukocyte integrin CD18 leading to recurrent bacterial infections. We report clinical features and analysis of neutrophil function from two mixed-breed canine littermates (one female and one male dog) similar to CLAD. The symptoms of pyogenic infection were first recognized at 3 months of age and since then the patients suffered from recurrent bacterial infections. These clinical findings were strongly suggestive of genetic phagocyte dysfunction. Neutrophil function tests revealed a marked reduction of serum-opsonized zymosan-mediated superoxide production in the two littermates. Neutrophils of the male dog revealed impaired integrin-mediated adherence and phagocytic activity, whereas ability of serum opsonization was normal. There was also a profound decrease of surface expression of CD11b/CD18 and beta2-integrin transcript level, detected by real-time RT-PCR without missense mutations unlike CLAD. Immunoblot analysis indicated that protein expression of cytochrome b(558) component gp91(phox), the cytosolic components p47(phox) and p67(phox) of NADPH oxidase components increased profoundly in the male. Our study suggests that decreased transcriptional levels of beta2-integrin without mutations, lead to downregulation of surface expression, resulting in multiple defects in adhesion-related neutrophil functions and consequently, recurrent bacterial infections from puppyhood.