The Vpu protein of human immunodeficiency virus type 1 (HIV-1) has been reported to enhance virion release from infected cells and to down-regulate the expression of CD4 on infected cells. Previous studies have shown that Vpu and the envelope glycoprotein precursor (gp160) are translated from different reading frames of the same bicistronic messenger RNA (mRNA). In order to assess the effect of the Vpu sequences 5' to the Env open reading frame on Env biosynthesis and pathogenesis, we have constructed a deletion mutant of a molecularly cloned chimeric simian--human immunodeficiency virus (SHIV(KU-1bMC33)) in which the entire coding region of vpu upstream of env had been deleted (novpuSHIV(KU-1bMC33)). While both SHIV(KU-1bMC33) and novpuSHIV(KU-1bMC33) synthesized comparable amounts of env mRNA in infected cells, the novpuSHIV(KU-1bMC33)-infected cells synthesized more Env precursor when standardized against the p57 Gag precursor protein. While more Env was synthesized than Gag in novpuSHIV(KU-1bMC33)-infected cells, pulse--chase analysis revealed that p27 Gag protein was released from infected cells with delayed kinetics, a reflection of the lack of a Vpu protein. Inoculation of novpuSHIV(KU-1bMC33) into two pig-tailed macaques resulted in no loss of circulating CD4(+) T cells. However, replicating virus could be detected in the lymphoid tissues (lymph nodes, spleen, thymus) 1 year after inoculation and the thymus of one of the macaques exhibited severe atrophy. The results of these studies indicate that the Vpu coding sequences upstream of Env may attenuate the level of Env precursor biosynthesis but significantly contribute to the pathogenesis of this SHIV in pig-tailed macaques.

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector-mediated gene therapy in GSD-Ia.

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.