Why does hiv have reverse transcriptase

It is composed of two different subunits, but both are encoded by the same gene. After the protein is made, one of the subunits is clipped to a smaller size shown in yellow here so that it can form the proper mate with one full-sized subunit shown in red.

Reverse transcriptase performs a remarkable feat, reversing the normal flow of genetic information, but it is rather sloppy in its job.

This is essential because they are the caretakers of our genetic information, and mistakes may be passed on to our offspring. Reverse transcriptase, on the other hand, makes lots of mistakes, up to about one in every 2, bases that it copies if this same error rate occurred in the "Molecule of the Month," there would be two typos in this month's installment. You might think that this would cause severe problems. But, in fact, this high error rate turns out to be an advantage for the virus in this era of drug treatment.

The errors allow HIV to mutate rapidly, finding drug resistant strains in a matter of weeks after treatment begins. Fortunately, the recent development of treatments that combine several drugs are often effective in combating this problem. Since the virus is simultaneously attacked by several different drugs, it cannot mutate to evade all of them at the same time.

Reverse transcriptase performs several different functions. This reaction is performed in the polymerase active site, which is formed by two sets of arms that surround the RNA and DNA. The polymerase site is at the top in this illustration, taken from PDB entry 2hmi. This is performed by a nuclease active site, which is located at the opposite end of the enzyme. This reaction is also performed by the polymerase site. The current collection of highly-effective drugs for fighting HIV infection are a major success of modern drug design.

Two types of drugs are used for blocking the action of reverse transcriptase and stopping HIV infection. One type is a modified nucleotide with a missing connector, such as the drug AZT. These are used by the enzyme like normal nucleotides and added to the growing chain. But, since they are missing a site for connecting the next nucleotide, the synthesis of the DNA chain is stopped.

The other type of drug binds on the back side of the enzyme and changes the shape of the active site, blocking its action. The drug Nevirapine, shown here in white from PDB entry 1jlb , is an example of this type of drug. To explore this structure in more detail, click on the accession code and choose one of the options for 3D viewing. The -ssDNA subsequently hybridises to the 3' terminus of the viral genome first strand transfer allowing negative strand DNA synthesis to continue [ 14 ].

Plus strand DNA synthesis is initiated and following a second strand transfer, double-stranded viral DNA is completed. The kinetics of HIV reverse transcription during virus replication has been analysed in several studies [ 13 — 17 ], including a synchronous one-step cell-cell HIV infection model used in our laboratory which shows distinct time delays in the appearance of -ssDNA 1.

Protein phosphorylation is known to regulate the enzymatic activity of a number of proteins including polymerases. Together, these results suggest that the RT process is activated during early infection, that RT is a substrate for phosphorylation and that phosphorylation may affect RT activity.

We therefore investigated whether HIV RT underwent post-translational modification, specifically phosphorylation, during the progression of a normal HIV infection. We report that RT p66 and p51 exist in virions and during HIV infection of cells as a number of protein isoforms, some of which are phosphorylated. The majority of RT is post-translationally modified and the major RT isoforms are present in HIV RTCs, suggesting that these isoforms play a biological function in the reverse transcription process inside the cell.

We firstly verified that our 2D gel electrophoresis system could accurately measure small changes in pI by determining the theoretical and experimental pIs of recombinant histidine tagged His -RT and GAPDH. These calculated pIs were greater than 2 pH units above the pKa of His and thus the His-tag would reduce the pI of either protein by only 0.

Additionally, we calculated the expected changes in pI for p66, p51 and GAPDH due to post-translational modification by phosphorylation or deamidation Table 1.

Other post-translational modifications such as acetylation could occur and would similarly induce an acidic shift in protein pI. A number of isoforms consistent in size with p66 or p51 were detected Figure 1 with the major isoforms present having pIs of 8.

The pIs of the most basic isoforms, p66 8. The pI difference between p66 8. These results are consistent with deamidation of both recombinant RT and GAPDH and demonstrate that changes in pI associated with post-translational modifications can be accurately measured using our 2D gel electrophoresis format.

RT isoforms are designated by black arrows and calculated pI indicated. As expected, two distinct bands corresponding to p66 and p51 were detected Figure 2A. The remaining sample was then analysed by 2D gel electrophoresis. Three distinct isoforms of p66 and p51 were identified Table 2. A summary of the reproducibly detected isoforms and potential post-translational modifications is presented in Table 3.

The isoforms of virion p66 8. The pIs of both of these major isoforms differed from that predicted for unmodified p66 8. The virion p51 isoforms showed a similar pI profile to the isoforms detected in recombinant RT, with the virion p51 8.

The pI values for p51 8. RT isoforms are present in purified HIV virions. RT was detected by Western blot using an anti-RT antibody. RT isoforms B are designated by black arrows and the calculated pI and expected position of p66 and p51 indicated. H3B cells thus represent a system to analyse changes in RT that occur co-incident with intracellular stimulation of reverse transcription and additionally offers the advantage of a synchronous and highly efficient infection model compared with a cell-free infection [ 13 ].

This allows high sensitivity in detecting RT protein. To analyse the RT in H3B producer cells we mixed H3B cells with uninfected Hut cells and immediately lysed cells prior to the opportunity for interaction, stimulation of RT or infection. Proteins were then immunoprecipitated and subjected to 2D gel electrophoresis. The two most abundant p66 8. New minor RT isoforms, not seen in virions were observed p66 8.

Minor differences in the p66 and p51 profiles were observed between these and the subsequently described experiments which are likely attributable to variation in HIV infection, immunoprecipitation efficiency, and sensitivity of western blot detection and spots that were variably observed are indicated on the figures with a white arrow.

A higher molecular weight RT immunoreactive species was sometimes observed eg Figure 3A, 3D which likely represents unprocessed Gag-Pol arising from the H3B producer cells. H3B and Hut cells were co-cultured for the indicated time period then lysed.

For panels A and B, lysates were immunoprecipitated using heat-inactivated AIDS patient sera cross-linked to protein A sepharose beads and washed. Minor differences in the p66 and p51 profiles were observed between experiments and spots not routinely observed are indicated by a white arrow. A H3B virus producer cells. H3B and Hut cells were co-cultured and lysed immediately. B Infected cell lysates. H3B and Hut cells were co-cultured and lysed at 40 min post-cell mixing.

The remainder of two selected fractions; D from the top of the gradient fraction 1 and E co-incident with the known sedimentation of RTCs fraction 5 , were TCA precipitated and subjected to 2D gel electrophoresis, as for panels A and B, above.

The same two major p66 8. However, the relative proportions of the major and minor isoforms differed, with the minor isoforms becoming more prominent and the major p66 8. Similar minor isoforms were present in these cells undergoing active reverse transcription compared with those detected in chronically infected virus producer H3B cells.

Infections were initiated by cell-cell mixing as previously, and after min, cell lysates were prepared and subjected to sucrose velocity gradient sedimentation.

This sedimentation technique was chosen since we have previously observed that it yields good separation of free protein fraction 1 and any remaining unactivated RT in pre-exisiting complexes from H3B cells fraction 7 from RTCs fraction 5 [ 2 , 26 ], the latter which we can monitor by virtue of the presence of newly synthesised reverse transcription products.

HIV reverse transcription products showed a peak in gradient fraction 5 1. Sucrose gradient fractions were then subjected to 2D gel electrophoresis and western blot for RT, as above. Fraction 1 from the top of the gradient and containing free protein showed RT isoforms with migration characteristics consistent with p66 8.

However, in fraction 5 containing RTCs, only isoforms with migration characteristics consistent with p66 8. Although this does not exclude the presence of other less abundant RT isoforms in RTCs that were not detected due to the much lower levels of RT protein present, our results confirm that the major isoforms of p66 8. As one of the most important forms of protein modification is phosphorylation, we analysed the susceptibility of RT isoforms to phosphatase treatment prior to 2D gel electrophoresis.

Next, HIV infection was initiated by mixing of H3B and Hut cells and after 40 min the cells were lysed and viral proteins immunoprecipitated. Precipitated proteins were divided equally and treated with or without calf intestinal alkaline phosphatase CIAP. The RT proteins were then analysed by 2D gel electrophoresis and detected by Western blot. The sample without phosphatase treatment showed a profile of p66 and p51 isoforms Figure 4A of calculated pI equivalent to p66 8. Some additional minor p66 and p51 isoforms were also observed, again highlighting the experimental variation in the minor RT isoforms.

Phosphatase treatment alters the RT isoforms detected. RT isoforms are designated by a black arrow and spots not routinely observed are indicated by a white arrow. Removal of phosphate groups should increase protein pI if phosphorylation is present. Phosphatase treatment clearly altered the observed p66 and p51 isoforms Figure 4B. The minor p66 isoforms, p66 8. This p66 8. Although most of p51 RT was relatively phosphatase resistant Figure 4B in one experiment phosphatase treatment reduced the levels of both p51 8.

We have previously observed variation in de-phosphorylation and that total de-phosphorylation of ovalbumin is time-dependent; indicating slow removal of certain phosphate groups CJ Bagley, unpublished results. Thus the variable susceptibly of some RT isoforms to de-phosphorylation may reflect reduced activity or restricted accessibility of the phosphatase enzyme to some phosphate groups present in the RT protein and thus we believe that p51 8.

Together the pI value and susceptibility to phosphatase treatment indicate that the RT isoforms p66 8. To analyse the significance of phosphorylated RT isoforms, cell lysates and virions were treated with or without phosphatase and RT activity was then assessed by in vitro exogenous RT activity assay Figure 5.

Since phosphatase itself could theoretically dephosphorylate dNTP's and influence the in vitro RT activity assay, we first validated measurement of RT activity in the presence of phosphatase and phosphatase buffering conditions.

RTCs were isolated by sucrose density gradient sedimentation, since this technique is best suited for concentrating particles into a more tightly sedimenting band than the velocity gradients used in Figure 3. Fractions 7—8, sedimenting at the previously defined density for RTCs [ 26 ] and containing newly synthesised reverse transcription products Figure 5B , were immunoprecipitated and subjected to dephosphorylation with CIAP, along with virions and cell lysates.

Dephosphorylation had no effect on the ability of RT found in virions, inside newly infected cells or associated with RTCs to perform in vitro reverse transcription Figure 5C. Additionally, other sources of phosphatase; Antarctic phosphatase and lambda phosphatase similarly had no effect on RT activity of virions data not shown , suggesting that phosphorylation makes limited contribution to the inherent activity of naturally occurring RT when measured in an in vitro assay.

Phosphatase treatment does not affect in vitro RT activity. Previous literature has suggested that RT may be subjected to post-translational modification, such as phosphorylation and it is well known that the process of reverse transcription is substantially activated upon cell infection.

We thus hypothesised that this activation of RT may be related to its post-translational modification, particularly phosphorylation. In this study we have shown by 2D gel electrophoresis that modified RT forms are the major RT protein present in virions, newly infected cells and RTC's. The same predominant RT isoforms with pI's of p66 8. The possibility that these represented an excess of inactive molecules present together with smaller levels of a modified active form, was considered unlikely since these forms predominated in semi-purified RTCs that are known to be supporting active reverse transcription.

The major RT isoforms observed corresponded to an undefined post-translational modification for p66 8. The major p51 8. Additionally, susceptibility of p51 8. Thus the major p51 8. This observed differential modification of p51 compared to p66 may be the result of i modification of a single p66 molecule of the RT homodimer that is then selectively targeted for cleavage giving rise to p51 and a mature RT heterodimer or ii selective modification of the p51 in the heterodimer post p66 cleavage.

The identification of these RT isoforms is novel. Previous studies have identified at least two isoforms of MA and CA [ 28 — 30 ] in HIV virions by 2D gel electrophoresis analysis followed by silver stain or western blot, but these studies have not identified isoforms of RT, possibly due to lower levels of RT or the use of isoelectric focussing strips of insufficient resolving power for the pI range of RT [ 30 , 31 ].

The RT isoforms we observed changed little between virus producer cells, virions and newly infected cells, although the minor RT isoforms became more abundant following infection. Some of the RT isoforms detected were phosphorylated, as suggested by their pI value and their susceptibility to dephosphorylation.

This is not surprising given our results showing that the major isoforms that would be present in samples from virions, infected cells and RTCs are p66 8.

Thus, naturally occurring phospho-RT isoforms are not a major contributor to RT activity, as measured in vitro , but could still be important for RT activity in the complex milieu of the infected cell, or may play a role in important structural interactions required for stability, movement and activity of the RTC intracellularly.

Conclusive analysis of the roles of phosphorylation at specific sites in the RT enzyme remain to be determined by mutagenesis of potential RT phosphorylation sites and analysis of subsequent 2D gel electrophoresis profiles. However, at present this kind of analysis is hampered by the reduced sensitivity for detection of RT following infection with cell-free virus and 2D gel analysis, as would be necessitated in these experiments.

In conclusion, we describe for the first time the presence of modified p66 and p51 RT isoforms and report that the same major p51 8. A better understanding of the post-translational modifications, the cellular enzymes involved and how these specifically influence RT activity inside the cell will be essential in elucidating the mechanisms for control of reverse transcription in newly infected cells.