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Alternative splicing plays a key role in the HIV-1 life cycle and is essential to maintain an equilibrium of mRNAs that encode viral proteins and polyprotein-isoforms. In particular, since all early HIV-1 proteins are expressed from spliced intronless and late enzymatic and structural proteins from intron containing, i.e. splicing repressed viral mRNAs, cellular splicing factors and splicing regulatory proteins are crucial for the replication capacity. In this review, we will describe the complex network of cis-acting splicing regulatory elements (SREs), which are mainly localized in the neighbourhoods of all HIV-1 splice sites and warrant the proper ratio of individual transcript isoforms. Since SREs represent binding sites for trans-acting cellular splicing factors interacting with the cellular spliceosomal apparatus we will review the current knowledge of interactions between viral RNA and cellular proteins as well as their impact on viral replication. Finally, we will discuss potential therapeutic approaches targeting HIV-1 alternative splicing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Employing antibodies against the cytoplasmic tail of the HIV-1 glycoprotein (Env-CT), in addition to gp160/gp41, we have identified several novel small Env proteins (<25kD) in HIV-1 transfected and infected cells. Mass spectrometric and mutational analyses show that two mechanisms contribute to their generation. Thus the protein, designated Tr-Env-CT (for truncated Env-CT), consists of the C-terminal 139 amino acids (aa) of Env (aa 718-856) with the N-terminal Q718 modified to pyroglutamic acid. It is likely derived from full-length Env protein by proteolytic processing. A further heterogeneous set of slightly larger proteins, termed Env-CT* species, are rather derived from spliced mRNAs containing only those Env C-terminal residues (aa 719-856) which overlap with the second tat and rev coding exons. They are N-terminally extended in the same reading frame. It is conceivable that essential Env-CT functions may be fulfilled by these novel species rather than by the full-length glycoprotein itself., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study focuses on the long stretch of highly conserved amino acids in the membrane proximal part of the HIV-1 cytoplasmic tail (Env amino acids (aa) 706-718) upstream of the overlap with the tat and rev second coding exons. Changes in Env aa 713 and 715, although they did not affect Env function, abrogated replicative spread. Other amino acid substitutions, i.e., 706-712, 714 and 716, despite their conservation, did not result in defective replicative phenotypes even in primary peripheral blood lymphocytes. Our results point to their involvement in presently unrecognized essential Env functions pertinent only in in vivo. Interestingly, changes in the codons for residues 717-718 as well as some mutations in residues 714-716 abrogated Gag expression but still allowed expression of functional Env in a rev-independent manner. This could be due to the inactivation of a rev-regulated negative element within the respective nucleotide sequence (8354-8368)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Usage of the HIV-1 major 5' splice site D1 is a prerequisite for generation of all spliced viral mRNAs encoding essential regulatory and structural proteins. We set out to determine whether flanking sequences ensure D1-activation. We found that an exonic splicing enhancer function is exerted by the region upstream of D1, which is crucially required for its activation. Additionally, we identified an intronic splicing regulatory element within the p17-instability element of the Gag-ORF enhancing D1-activation. Furthermore, our experimental data demonstrated that sequence motifs displaying high similarity to consensus binding sites for SR protein SC35 (SRSF2) overlapping with D1 fine-tune its activation. Our results reveal that D1-activation is safe-guarded by the interplay of upstream and downstream located splicing enhancer elements ensuring usage of D1 even if its strength is decreased upon mutation. The identification of sequence elements activating D1-usage sheds further light on the balanced expression of alternatively spliced HIV-1 mRNAs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The HIV-1 Vpu and Env proteins are translated from 16 alternatively spliced bicistronic mRNA isoforms. Translation of HIV-1 mRNAs generally follows the ribosome scanning mechanism. However, by using subgenomic env expression vectors, we found that translation of glycoprotein from polycistronic mRNAs was inconsistent with leaky scanning. Instead a conserved minimal upstream open reading frame (uORF) consisting only of a start and stop codon that overlaps with the vpu start site, appears to augment access to the env start codon downstream. Mutating the translational start and stop codons of this uORF resulted in up to fivefold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter Env expression.

The human immunodeficiency virus type 1 (HIV-1) uses an elaborate alternative splicing pattern for the generation of both the 1.8-kb as well as the 4-kb classes of mRNA. An additional diversity of transcripts in both classes is created by the optional inclusion of the small exons 2 and 3 in the leader sequence. To analyze a possible influence of these leader exons on HIV-1 gene expression, several series of expression vectors with different leaders were constructed, expressing either Rev and Env or a heterologous coding sequence, i.e., the chloramphenicol acetyl transferase (CAT) ORF. Transfection experiments of HeLa-T4(+) cells revealed for all series of constructs that mRNA as well as protein expression was stimulated by the presence of exon 2 and reduced by exon 3. The function of the leader exons 2 and 3 is neither dependent on the regulatory proteins Tat or Rev nor on viral coding sequences. Neither transcription rates nor stability of polyadenylated RNAs were found to be responsible for the different levels of steady-state mRNA. When either exon 2 or 3 was inserted into a heterologous intron, processing of the primary transcripts generated identical mRNA species while maintaining the differences in exon 2/3-dependent mRNA steady-state levels. These results may be explained by exon-specific nuclear RNA degradation rates, as also indicated by results from an in vitro degradation assay using a HeLa nuclear extract., (Copyright 2001 Academic Press.)