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Journal of Virology, February 2008, p. 1600-1604, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.02295-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Gag-Processing Defect of Human Immunodeficiency Virus Type 1 Integrase E246 and G247 Mutants Is Caused by Activation of an Overlapping 5' Splice Site{triangledown}

Dibyakanti Mandal, Zehua Feng, and C. Martin Stoltzfus*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 22 October 2007/ Accepted 5 November 2007


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ABSTRACT
 
We have previously described several human immunodeficiency virus type 1 (HIV-1) mutants that are characterized by an excessive-RNA-splicing phenotype and reduced virus particle production. In one of these mutants (NLD2up), the sequence of 5' splice site D2 was changed to a consensus splice donor site. This splice site overlaps the HIV-1 integrase reading frame, and thus, the NLD2up mutant also bears a G-to-W change at amino acid 247 of the integrase. A previously described E-to-K mutant at position 246 of the C-terminal domain of the integrase, which resulted in a G-to-A mutation at the +3 position of overlapping splice donor D2 (NLD2A3), was also shown to affect virus particle production and Gag protein processing. By using second-site mutations to revert the excessive-splicing phenotype, we show that the effects on Gag protein processing and virus particle production of both the NLD2up and NLD2A3 mutants are caused by excessive viral RNA splicing due to the activation of the overlapping 5' splice site and not to the changes in the integrase protein. Both integrase protein mutations, however, are lethal for virus infectivity. These studies suggest that changes in the usage of overlapping splice sites may be a possible alternative explanation for a defective virus phenotype resulting from changes in protein-coding sequences or in the nucleotide sequence during codon optimization.


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TEXT
 
Human immunodeficiency virus type 1 (HIV-1) RNA splicing is inefficient, and approximately half of HIV-1 mRNA remains unspliced (US); the remainder of the mRNA is incompletely spliced (IS) to messages for Env, Vpu, Vif, and Vpr or completely spliced to messages for Tat, Rev, and Nef. US mRNA, which is transported to the cytoplasm by a Rev-dependent process, encodes messages for viral Gag and Gag-Pol genes and also serves as genome RNA for packaging into virions. In addition, some of the mRNAs include small noncoding exons (exon 2 or exon 3), which are derived from sequences in the integrase and Vif reading frames, respectively. More than 40 unique alternatively spliced HIV-1 mRNAs are produced by utilization of four donor splice sites (5' SS) and eight viral acceptor splice sites (3' SS) [Fig. 1A]. HIV-1 alternative splicing is regulated by the presence of suboptimal 5' SS and 3' SS, which decrease the affinity of cellular factors to the splicing sites, as well as by exonic splicing enhancers and exonic/intronic splicing silencers, which represent high-affinity binding sites for cellular factors that either promote or inhibit splicing at nearby splice sites (for a recent review of HIV-1 splicing, see reference 15).


Figure 1
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  		FIG. 1. Viral RNA produced within HIV-1-infected cells and virus mutants used in this study. (A) (i) HIV-1 genes are shown relative to the long terminal repeats (LTRs). (ii) 5' SS and 3' SS located within the 9-kb HIV-1 genome (D1 to D4 and A1 to A7, respectively) are shown. (iii) Completely spliced and IS HIV-1 mRNAs (~1.8 kb and ~4 kb, respectively) are depicted as black boxes. Spliced mRNAs are denoted by the translated open reading frames and by the exon content. The IS mRNAs, denoted with an "I," are differentiated from completely spliced mRNAs by inclusion of the intron between 5' SS D4 and 3' SS A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) are included within some of the 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I). (B) HIV-1 5' SS D2 mutants used in this study are shown. The sequences are aligned and compared to the consensus metazoan splice donor sequence. 5' SS D2 is indicated by an arrow. "r" at the +3 position in the consensus sequence can be either "g" or "a." Amino acid sequence changes within the overlapping integrase protein reading frame are also indicated. WT, wild type. Uppercase letters represent exon 2 sequences, and lowercase letters represent intron sequences. The underlined sequences represent integrase codons 246 and 247.

We have previously shown that 5' SS D2 is suboptimal. When this splice site was mutated (underlining) to the metazoan consensus 5' SS signal (G/GUGAAG to G/GUGAGU) in order to increase its affinity for cellular U1 snRNA, splicing at 3' SS A1 was dramatically increased, the accumulation of US RNA was decreased, and production of virions was decreased approximately 90% (11). Increased splicing at 3' SS A1 is a consequence of exon definition, in which recognition of an upstream 3' SS by the cellular splicing machinery can be strengthened by the downstream 5' SS bordering the exon (5, 8, 13). The D2up mutation, in addition to the effect on splicing at 3' SS A1, creates a glycine-to-tryptophan change at amino acid residue 247 of the overlapping integrase reading frame (Fig. 1B). Lu et al. previously reported that a glutamic acid-to-lysine change at the adjacent amino acid residue 246 resulted in a defect in Gag processing and virus particle production that was unique among all the C-terminal integrase mutants tested in the study (10). However, as shown in Fig. 1B, this mutation also changes the overlapping 5' SS D2 sequence from G/GUGAAG to G/GUAAAG (Fig. 1B). For a weak 5' SS with a nonconsensus A at position +5, an A at position +3 is preferred, presumably because of the increased stability of an A-U base pair compared to a G-U base pair (6). Thus, it was not clear whether the observed protein-processing defects and reduced particle production of the G247W and E246K mutants were caused by activation of 5' SS D2 or to changes in the activity of the integrase protein.

To distinguish these two possibilities, we generated second-site mutations of the G247W (D2up) and E246K (D2A3) mutants that acted to inhibit splicing at 5' SS D2 by weakening its affinity for U1 snRNA (Fig. 1B). To this end, second-site mutations were created within the GU sequence, which is conserved in more than 99% of all mammalian 5' SS. Because U within the GU sequence is present at the wobble position of the integrase glycine codon (GGU), it was possible to interfere with splicing at 5' SS D2 without altering the overlapping integrase protein sequence. We then compared the properties of the second-site mutants to those of the single mutants.

We first tested the abilities of the second-site mutations to inhibit splicing at 5' SS D2 by performing reverse transcriptase (RT) PCR analysis of total RNA isolated from 293T cells transfected with the infectious HIV-1 plasmid pNL4-3 (2) and mutated variants of the plasmid. Figure 2A shows that most of the HIV-1 1.8-kb mRNA species in pNLD2up-transfected cells included the 49-nt exon (exon 2) between A1 and D2, as indicated by the absence of mRNA species 1.4.7, 1.4a/b.7, and 1.5.7 and increased amounts of species 1.2.4.7, 1.2.4a/b.7, 1.2.3.5.7, and 1.2.5.7. Second-site mutations of GU to either GA or GG (pNLD2GAup and pNLD2GGup) prevented exon 2 inclusion, as indicated by the absence of detectable mRNA species containing exon 2. A GU-to-GC second-site mutation (pNLD2upGC) reduced but did not block inclusion of exon 2. Previous results indicated that approximately 0.5% of mammalian 5' SS contain a noncanonical GC rather than GU; most of these 5' SS follow consensus at the other positions of the splice site (1, 7, 16). The pNLD2A3 mutant also demonstrated increased inclusion of exon 2 (Fig. 2A). The extensive inclusion of exon 2 in pNLD2A3 mRNA was completely abrogated by a GU-to-GC second-site mutation (Fig. 2A, mutant pNLD2GCA3). Thus, excessive splicing at 5' SS D2 was inhibited by appropriate second-site mutations within the conserved GU sequence of the splice site.


Figure 2
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  		FIG. 2. Analysis of spliced and US HIV-1 RNAs produced in cells transfected with wild-type and mutant HIV-1 plasmids. (A) Total-RNA samples from 293T cells transfected with the indicated plasmids were analyzed by RT-PCR for completely spliced 1.8-kb HIV-1 mRNA (12). The bands corresponding to viral mRNA species are indicated. (B) Northern blot analysis of total RNA isolated from cells transfected with NL4-3 and 5' SS D2 mutant plasmid was performed using a 32P-labeled probe from exon 7 that detects all HIV-1 viral-mRNA species (12). The positions of US, IS, and completely spliced (CS) products are indicated. The position of vif mRNA is also shown. (C) vif mRNA produced in the cells transfected with pNL4-3 or the indicated 5' SS D2 mutant plasmids was determined by quantitative real-time PCR. PCR amplification products were detected using SYBR green. The amounts of vif mRNA are expressed relative to that of pNL4-3. The error bars indicate standard deviations.

We next determined the relative levels of US, IS, and completely spliced mRNAs by Northern blot analysis. As previously reported (11), a greatly reduced level of US mRNA and an increased level of IS mRNA compared to the wild type were present in pNLD2up-transfected cells (Fig. 2B). Most of the IS mRNA migrated more slowly than the major wild-type IS mRNA band, corresponding to a size expected for vif mRNA, which is produced by splicing from 5' SS D1 to 3' SS A1. The GU second-site mutants pNLD2GAup and pNLD2GGup and, to a lesser extent, pNLD2GCup demonstrated increases in the relative amounts of US RNA and decreases of IS mRNA. In pNLD2A3-transfected cells there was also increased IS vif mRNA relative to US mRNA, but at a reduced level compared to pNLD2up-transfected cells (Fig. 2B). In cells transfected by the second-site GU mutant pNLD2GCA3 there was a decrease in IS viral mRNA relative to US viral mRNA. These results indicated that the excessive-splicing phenotypes of both the D2up and D2A3 mutations were abrogated by the GU second-site mutations.

To further confirm that the second-site changes in 5' SS D2 caused reductions in splicing at 3' SS A1, we determined the levels of vif mRNA in cells transfected with the wild-type and mutant plasmids by quantitative real-time PCR. In pNLD2up- and pNLD2A3-transfected cells there were approximate 25-fold and 5-fold increases, respectively, in vif mRNA levels. In contrast, the second-site GU mutations pNLD2GAup, pNLD2GGup, and pNLD2GCA3 demonstrated reduced levels of vif mRNA compared to wild-type pNL4-3. We attribute this reduction to reduced base pairing of U1 snRNA to the mutated compared to the wild-type 5' SS D2, resulting in reduced splicing at 3' SS A1, as predicted by the exon definition hypothesis. Mutant pNLD2GCup demonstrated a somewhat elevated vif mRNA level compared to the wild type. This increased usage of 3' SS A1 is consistent with increased levels of mRNA species 1.2.4.7 compared to 1.4.7, as seen in Fig. 2A (compare pNL4-3 to pNLD2GCup).

We then determined whether the GU second-site mutations could rescue virus particle production by measuring the levels of RT activity in the media of transfected cells (Fig. 3A). The particle production levels of the NLD2up and NLD2A3 mutants were reduced to approximately 10% and 20% of wild type, respectively. Second-site GU mutations to GC, GA, and GG within NLD2up and the GU-to-GC second-site mutation within pNLD2A3 restored virus particle production to approximately wild-type levels. Particle production was also compared to the single mutations at the +2 position (pNLD2GC, pNLD2GA, and pNLD2GG), and no significant effect on particle production was observed. Since the second-site mutations do not change the protein sequence of the integrase protein, we concluded that the reduced virus particle production exhibited by pNLD2up and pNLD2A3 was a consequence of an excessive-RNA-splicing phenotype.


Figure 3
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  		FIG. 3. Analysis of HIV-1 particle production and protein processing of wild-type and 5' SS D2 mutants. (A) RT activities of cell supernatants from cells transfected with the indicated 5' SS D2 mutant cells were determined as described previously (17). The RT activities of the 5' SS D2 mutants are expressed as fractions of that of wild-type pNL4-3. The error bars indicate standard deviations. (B) Proteins from cell lysate, cell medium, and 10-fold-diluted medium of cells transfected with the indicated plasmids were separated on a 10% polyacrylamide gel electrophoresis gel, and HIV-1 proteins were detected by immunoblotting using anti-p24 antibody obtained from the NIH AIDS Research and Reference Reagent Program. For the internal loading controls, beta-tubulin proteins present in the cell lysates were detected using anti-tubulin antibody (E7) obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa.

We then compared the levels of viral proteins isolated from the cells and media by Western blotting using an anti-p24 antibody (Fig. 3B). In cell lysates, mutants pNLD2up and pNLD2A3 both demonstrated increased ratios of the Gag precursor Pr55 to the processing intermediate Pr41 and the final product p24 compared to the wild type. There were also reduced levels of p24 present in the medium, which was consistent with the decreased particle production determined by the RT assays shown in Fig. 3A. In contrast, the second-site GU mutants demonstrated wild-type ratios of Gag precursor to product and wild-type levels of p24 in the medium. We concluded from these results that the Gag-processing defects of pNLD2up and pNLD2A3 were caused by excessive RNA splicing and not by changes in the integrase protein sequence.

Finally, we tested mutant viruses generated by transient transfection for infectivity in LuSIV CEMx174 cells (14). The results shown in Fig. 4 indicated that the GU single mutations at the +2 position had little or no effect on virus infectivity. This was expected, since there were no changes in the integrase amino acid sequence. The small decreases in the infectivities of the GU single mutations compared to NL4-3 may be due to small changes in the splicing balance caused by mutation of 5' SS D2. On the other hand, the NLD2up and NLD2A3 virus mutants, as well as the NLD2up and NLGCA3 second-site GU mutants, were equally noninfectious in this assay. These results suggest that both the E246K and G247W mutations affect integrase function, since the failure to infect the LuSIV cells is independent of the excessive-RNA-splicing and protein-processing phenotypes. Our results are consistent with previous results of Lu et al., which indicated that the HIV-1E246K mutant virus was replication defective in a single-round infectivity assay (10).


Figure 4
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  		FIG. 4. Infectivities of NL4-3 and 5' SS D2 mutant viruses. LuSIV cells were infected for 36 h with equivalent cell supernatant RT activities of 293T cells transfected with each of the indicated virus plasmids. Cell lysates from the infected LuSIV cells were used to determine luciferase activities (14). Uninfected cell lysate was used as a negative control (Mock). The error bars indicate standard deviations.

Our data clearly show that mutations within the HIV-1 integrase coding sequence (E246K and G247W), while at positions distant from the Gag and Pro sequences, affect Gag protein processing by activating viral RNA splicing. The nucleotide sequence of the HIV-1 integrase reading frame in the region overlapping 5' SS D2 is highly conserved in different HIV-1 strains, and our results suggest that this conservation arises because of the necessity for HIV-1 to maintain an optimal amount of RNA splicing, as well as integrase function. Our results further suggest that alterations in usage of overlapping splice sites should be considered a possible alternative explanation for defective virus phenotypes due to changes in protein-coding sequences.

How does excessive splicing of the NLD2up and NLD2A3 mutants cause Gag-processing/virus particle production defects? One possible explanation is that overproduction of Vif protein may affect Gag protein processing by inhibiting HIV-1 protease function (3). However, we believe that this is not the case, since we found that processing of Gag protein was similarly defective in cells transfected by pNLD2up or by a Vif-minus variant of pNLD2up (data not shown). Another possible explanation is that particle assembly is affected by the reduced amount of US viral RNA available for packaging into virions. This seems unlikely, since normal levels of virus particles have been shown to be produced by HIV-1 mutants that do not specifically package viral RNA (4, 9). We showed above that Gag proteins in particles produced by NLD2up and NLD2A3 contain p24, indicating that mutant viral protease has normal activity. These particles also have RT activity. Thus, we believe that the most likely explanation for the results is that reduced US RNA levels result in reduced translation of Gag, reduced intracellular levels of Gag, and consequent inefficient virus assembly.


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ACKNOWLEDGMENTS
 
HIV-1-related reagents were supplied by the NIH AIDS Research and Reference Reagent Program. Monoclonal antibody E7 was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa.

This research was supported by PHS grant AI36073 from the National Institute of Allergy and Infectious Diseases.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7793. Fax: (319) 335-9006. E-mail: marty-stoltzfus{at}uiowa.edu Back

{triangledown} Published ahead of print on 21 November 2007. Back


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Journal of Virology, February 2008, p. 1600-1604, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.02295-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mandal, D., Exline, C. M., Feng, Z., Stoltzfus, C. M. (2009). Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5' Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G. J. Virol. 83: 6067-6078 [Abstract] [Full Text]  
  • Exline, C. M., Feng, Z., Stoltzfus, C. M. (2008). Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression. J. Virol. 82: 3921-3931 [Abstract] [Full Text]  

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