Abstract
In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.
Highlights
From a genome of only 9000 nt in length, Human Immunodeficiency Virus type 1 (HIV-1) directs the synthesis of 15 proteins essential for its replication and dissemination
Because Tat2 and Tat6 required splicing at site A1 and Vpr1, Vpr3 and Tat3 mRNAs at site A2, we concluded that ASF/SF2 participated in a positive regulation of splicing at sites A1 and A2, while SC35 and 9G8 preferentially enhanced splicing at site A3 necessary for Tat mRNA synthesis (Figure 1)
These results are in agreement with those obtained in HeLa cells using a truncated non-infectious HIV-1 DNA construct [26] and showed that SR proteins profoundly changed the HIV-1 splicing pattern
Summary
From a genome of only 9000 nt in length, HIV-1 directs the synthesis of 15 proteins essential for its replication and dissemination (for review see ref. [1]). From a genome of only 9000 nt in length, HIV-1 directs the synthesis of 15 proteins essential for its replication and dissemination In order to generate mRNAs required for the synthesis of these proteins, HIV-1 uses the cellular splicing machinery. The 2 kb mRNAs are fully spliced and principally encode the regulatory proteins Tat and Rev and accessory proteins Nef and Vpr. The single-spliced 4 kb RNAs are bicistronic and code for the Env glycoproteins and viral factor Vpu, and the unspliced 9 kb RNA serves both as mRNAs for the (page number not for citation purposes). A3A4c A5 pol vpr rev vif tat vpu D2 D3 D4
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