HIV-1 accessory proteins: Vpu and Vif.

HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins.

An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA.

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

Quantitation of parvalbumin+ neurons and human immunodeficiency virus type 1 (HIV-1) regulatory gene expression in the HIV-1 transgenic rat: effects of vitamin A deficiency and morphine.

Vitamin A (VA) deficiency in human immunodeficiency virus (HIV) infection has been associated with more progressive HIV disease, which may be enhanced by opioid use. In these studies, we examined the effects of VA deficiency and morphine on frontal cortex neuronal numbers in the HIV-1 transgenic (Tg) rat. These studies showed that total numbers of neurons were similar for rats on the VA-deficient diet as for rats on the normal diet and these numbers were not affected by treatment with morphine. In contrast, numbers of neurons that expressed the calcium-binding protein parvalbumin, which is a marker interneurons that express the inhibitory neurotransmitter gamma-aminobutyric acid (GABAergic neurons) were decreased for wild-type (Wt) rats on the VA-deficient diet and for Wt rats treated with morphine. In addition, parvalbumin+ neurons were also decreased for Tg rats on a normal diet but increased to normal levels when these animals were placed on the VA-deficient diet and treated with morphine. Analysis of expression of the genes that code for the HIV regulatory proteins vif, vpr, nef, and tat in frontal cortex and adjacent subcortical white matter showed that tat expression was increased in the morphine-treated Tg rat on the VA-deficient diet as compared to untreated Tg rats on the normal diet and untreated VA-deficient rats. These studies therefore suggest that VA deficiency, opioid exposure, and HIV infection alone and in combination may potentially alter neuronal metabolic activity and induce cellular stress, resulting in the observed changes in levels of parvalbumin expression. The specific mechanisms that underlie these effects require further study.

Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.