Transframe Protein Research Articles

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport.

Programmed ribosomal frameshifting is a translational recoding phenomenon in which a proportion of ribosomes are stimulated to slip backwards or forwards on an mRNA1, rephasing the ribosome relative to the mRNA. While frameshifting is often employed by viruses2, very few phylogenetically conserved examples are known in vertebrate genes and the evidence for some of these is controversial3,4. Here we report a +1 frameshifting signal in the coding sequence of the human gene PLEKHM2, encoding the ARL8-dependent, lysosome-kinesin-1 adaptor protein PLEKHM25. This +1 frameshifting signal, UCC_UUU_CGG, is highly conserved in vertebrates and exhibits an influenza virus-like frameshift motif with similar efficiency6,7. Purification and mass spectrometry of GFP-tagged trans-frame protein from cells confirms frameshifting. Structure prediction shows that the new C-terminal domain generated by this frameshift forms an alpha-helix. This additional domain relieves PLEKHM2 from autoinhibition, allowing it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Thus, the frameshift proteoform generates a constitutively active adaptor of kinesin-1.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Ablation of Programmed -1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity.

Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. -1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

Soybean Golgi SNARE 12 protein interacts with Soybean mosaic virus encoded P3N-PIPO protein

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is one of the most prevalent and devastating viral pathogens in soybean-growing regions worldwide. It is generally accepted that symptom development of a viral plant disease results from molecular interactions between the virus and its host plant. P3N-PIPO, as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO of the Potyvirus, plays a key role of viral cell-to-cell movement. This study provides evidence that Golgi SNARE 12 (designated as GOS12) protein of soybean interacts with SMV P3N-PIPO via a two-hybrid yeast system by screening a soybean cDNA library. Bimolecular fluorescence complementation (BiFC) assay further confirmed the interaction, which occurred in the cytomembrane of Nicotiana benthamiana cells. We also confirmed that the domain involved in the interaction is PIPO domain of P3N-PIPO by two-hybrid yeast system and BiFC. It is possible that the GOS12 is an essential host factor for PD targeting of P3N-PIPO protein of potyvirus.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus.

The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Transactivation of programmed ribosomal frameshifting by a viral protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement

A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.

Evidence for ribosomal frameshifting and a novel overlapping gene in the genomes of insect-specific flaviviruses

Flaviviruses have a positive-sense, single-stranded RNA genome of ∼11 kb, encoding a large polyprotein that is cleaved to produce ∼10 mature proteins. Cell fusing agent virus, Kamiti River virus, Culex flavivirus and several recently discovered flaviviruses have no known vertebrate host and apparently infect only insects. We present compelling bioinformatic evidence for a 253–295 codon overlapping gene (designated fifo) conserved throughout these insect-specific flaviviruses and immunofluorescent detection of its product. Fifo overlaps the NS2A/NS2B coding sequence in the − 1/+ 2 reading frame and is most likely expressed as a trans-frame fusion protein via ribosomal frameshifting at a conserved GGAUUUY slippery heptanucleotide with 3′-adjacent RNA secondary structure (which stimulates efficient frameshifting in vitro). The discovery bears striking parallels to the recently discovered ribosomal frameshifting site in the NS2A coding sequence of the Japanese encephalitis serogroup of flaviviruses and suggests that programmed ribosomal frameshifting may be more widespread in flaviviruses than currently realized.

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6(gag) sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3' end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6(gag) reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

Influence of extended mutations of the HIV-1 transframe protein p6⁎ on Nef-dependent viral replication and infectivity in vitro

The HIV-1 transframe protein p6⁎ known to modulate HIV-1 protease activation has been suggested to interact with the viral pathogenicity factor Nef. However, a potential interaction site in p6⁎ has not been mapped so far. To evaluate effects of p6⁎ modification on viral replication in light of Nef function, clustered substitutions were introduced into the central p6⁎ region of the infectious provirus NL4-3 and virus growth and composition of the various mutants was analyzed in different cell cultures in the presence or absence of Nef. Whereas clustered p6⁎ substitutions did neither affect particle incorporation of Nef, nor precursor maturation or viral infectivity, a simultaneous substitution of 40 of the total 56 p6⁎ residues significantly diminished viral infectivity and replication in a Nef-independent manner. Furthermore, this extended modification was not capable of rescuing the negative effects of a transdominant Nef mutant on particle production suggesting that the proposed target for Nef interaction in Gag-Pol is located outside the modified p6⁎ region. In sum these data strongly argue against a functional connection of the central p6⁎ region and Nef during viral life cycle.

Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation

The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.

Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma

BackgroundThe genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood.ResultsWe report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species.ConclusionOur results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.

Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing

The proteolytic processing of human immunodeficiency virus (HIV) particles mediated by the viral pol-encoded protease (PR) is essential for viral infectivity. The pol coding sequence partially overlaps with the gag coding sequence and is translated as a Gag-Pol polyprotein precursor. Within Gag-Pol, the C-terminal p6(gag) domain is replaced by a transframe peptide referred to as p6*, which separates the Gag nucleocapsid domain from PR. Several previous in vitro studies have ascribed a PR-suppression regulatory function to p6*. Here, it was demonstrated that an HIV-1 Gag-Pol lacking p6* is efficiently incorporated into virions when coexpressed with HIV-1 Gag precursor. However, the released virions are not processed appropriately and show a greatly reduced viral infectivity. This suggests that the p6* is indispensable during the process of PR-mediated virus particle maturation.

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-C(nn) (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, C(nn) at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32 degrees C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme.

Involvement of two domains with helix-turn-helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats.

The insertion element IS1 has two open reading frames (ORFs), insA and insB, and produces a transframe protein InsAB, known as IS1 transposase, by translational frameshifting. The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS1 transposition. Unless frameshifting occurs, IS1 produces InsA protein, which also binds to IRs and therefore acts as an inhibitor of transposition, as well as a transcriptional repressor of the promoter in IRL. A helix-turn-helix (HTH) motif present in both transposase and InsA is thought to be involved in IR-specific DNA binding. A comparison of transposases encoded by IS1 family elements reveals that the N-terminal regions contain four conserved cysteine residues, which appear to constitute a C(2)C(2) zinc finger (ZF) motif. This motif is also thought to be involved in IR-specific DNA binding. In this study, we show that IS1 transposases with an amino acid substitution in the HTH or ZF motif lose the ability to promote transposition. We also show that transposases, as well as InsA proteins with the same substitution, lose the ability to repress the activity of the IRL promoter, and that purified InsA mutant proteins lose the ability to bind to the IRL-containing fragment. Furthermore, we show that InsA protein co-ordinates Zn(II) with the four cysteine residues as ligands and loses the ability to bind to the IRL-containing fragment in the presence of an agent chelating Zn(II). These findings indicate that IS1 transposase has two domains with HTH and ZF motifs responsible for IR-specific DNA binding in promoting transposition. It is assumed that the two domains are needed for transposase to bind to each IR in an oriented manner in order to place a catalytic domain in the C-terminal region of the transposase to a region around the IR end, where the strand transfer reaction occurs in a transpososome.

Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant.

The protease (PR) from human immunodeficiency virus (HIV) is essential for viral replication: this aspartyl protease, active only as a dimer, is responsible for cleavage of the viral polyprotein precursors (Gag and Gag-Pol), to release the functional mature proteins. In this work, we have studied the structure-function relationships of the HIV PR by combining a genetic test to detect proteolytic activity in Escherichia coli and a bacterial two-hybrid assay to analyze PR dimerization. We showed that a drug-resistant PR variant isolated from a patient receiving highly active antiretroviral therapy is impaired in its dimerization capability and, as a consequence, is proteolytically inactive. We further showed that the polypeptide regions adjacent to the PR coding sequence in the Gag-Pol polyprotein precursor, and in particular, the transframe polypeptide (TF), located at the N terminus of PR, can facilitate the dimerization of this variant PR and restore its enzymatic activity. We propose that the TF protein could help to compensate for folding and/or dimerization defects in PR arising from certain mutations within the PR coding sequence and might therefore function to buffer genetic variations in PR.

The mature reverse transcriptase molecules in virions of mouse mammary tumor virus possess protease-derived sequences

Our efforts to express in bacteria the enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV) have shown that the RT is active only after adding 27 amino acid residues, which are derived from the end of the pro gene, to the amino-terminus of the RT (Biochem, J. (1998) 329, 579–587). In the present study we have tested whether the mature RT found in virions is also fused to protease-derived sequences. To this end, we have analyzed the RT molecules in virions of MMTV by using two antisera directed against peptides, derived from either the carboxyl-terminus of MMTV protease or the middle of MMTV RT. The data suggest that the mature RT, located in virions, contains at its amino-terminus sequences from the carboxyl-terminus of the protease protein. This finding supports previous suggestions that MMTV RT is a transframe protein (derived from both pro and pol reading frames of MMTV) and that amino acid residues located at the carboxyl-terminus of the protease have a dual usage as integral parts of both the protease and the RT enzymes.

A novel protease processing site in the transframe protein of human T-cell leukemia virus type 1 PR76(gag-pro) defines the N terminus of RT.

The genomic RNA of human T-cell leukemia virus type 1 encodes three polyproteins, Gag, Gag-Pro, and Gag-Pro-Pol, which are translated as a result of no, one, and two frameshifts, respectively. In this report we demonstrate that the 77 residues encoded at the C terminus of the Gag-Pro precursor can be collectively detected as an 8-kDa transframe protein (TFP) in virions. Mutant viruses with a C-terminally truncated TFP (19, 32, or 50 residues) had essentially a wild-type phenotype in vitro. However, a virus mutant that encoded only the Gag and Gag-Pro-Pol polyproteins due to a mutation in the second frameshift site, and hence did not produce TFP, was noninfectious. Mutation analysis of the proteolytic cleavage site between PR and TFP revealed the presence of an additional site and the existence of a p1 peptide separating protease and TFP. While removal of the cleavage site at the PR-p1 junction had a modest effect on virus replication, mutation of the p1-TFP cleavage site led to noninfectious virus and the loss of reverse transcriptase activity. Determination of the amino-terminal sequence of a hemagglutinin-tagged RT demonstrated that the same site is used in processing the Gag-Pro-Pol precursor and thus defines the start of mature RT. Neither mutation alone or in combination caused changes in the amounts or processing patterns of the Gag polyprotein, indicating that protease is active independent of its C terminus.

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic RNA dimer stability.

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.