Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

Boyer, Paul L.; Stenbak, Carolyn R.; Clark, Patrick K.; Linial, Maxine L.; Hughes, Stephen H.

doi:10.1128/jvi.78.12.6112-6121.2004

Cited by 28 publications

(40 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1) (9). After cleavage of Pol, free IN is active (30), and PR-RT has very high levels of reverse transcriptase and RNase H activity (2,29). It is possible that weak dimers of PR-RT, present at a low level in virions, account for the report of low levels of PFV Gag cleavage in newly infected cells (20).…”

Section: C-terminal Deletion Of the In Domain Disrupts Pr Proteolyticmentioning

confidence: 99%

Foamy Retrovirus Integrase Contains a Pol Dimerization Domain Required for Protease Activation

Lee¹,

Roy²,

Jackson³

et al. 2011

J Virol

Self Cite

View full text Add to dashboard Cite

Section: C-terminal Deletion Of the In Domain Disrupts Pr Proteolyticmentioning

confidence: 99%

Foamy Retrovirus Integrase Contains a Pol Dimerization Domain Required for Protease Activation

Lee¹,

Roy²,

Jackson³

et al. 2011

J Virol

Self Cite

View full text Add to dashboard Cite

“…The RNase H assay has been previously described (6). The RNA oligonucleotide was obtained from Dharmacon Research, Inc., and has the sequence 5=-GGGCCACUUUUUAAAAGAAAAGGGGGGA CUGGAAGGGCUAAUUCACUCAC-3=.…”

Section: Preparation Of Hiv-1 Rtmentioning

confidence: 99%

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

Boyer

Clark

Hughes

2012

J Virol

Self Cite

View full text Add to dashboard Cite

bAs anti-HIV therapy becomes more widely available in developing nations, it is clear that drug resistance will continue to be a major problem. The related viruses HIV-1 and HIV-2 share many of the same resistance pathways to nucleoside reverse transcriptase inhibitors (NRTIs). However, clinical data suggest that while HIV-1 reverse transcriptase (RT) usually uses an ATP-dependent excision pathway to develop resistance to the nucleoside analog zidovudine (AZT), HIV-2 RT does not appear to use this pathway. We previously described data that suggested that wild-type (WT) HIV-2 RT has a much lower ability to excise AZT monophosphate (AZTMP) than does WT HIV-1 RT and suggested that this is the reason that HIV-2 RT more readily adopts an exclusion pathway against AZT triphosphate (AZTTP), while HIV-1 RT is better able to exploit the ATP-dependent pyrophosphorolysis mechanism. However, we have now done additional experiments, which show that while HIV-1 RT can adopt either an exclusion-or excision-based resistance mechanism against AZT, HIV-2 RT can use only the exclusion mechanism. All of our attempts to make HIV-2 RT excision competent did not produce an AZT-resistant RT but instead yielded RTs that were less able to polymerize than the WT. This suggests that the exclusion pathway is the only pathway available to HIV-2. HIV-1 infection has been the target of various multidrug therapies, but to date, the effectiveness of all anti-HIV drugs has been blunted by drug-resistant mutations that arise in the genome of the virus. Reverse transcriptase (RT) is an enzyme that contains two enzymatic activities, a DNA polymerase that can copy either an RNA or DNA template, and an RNase H, which degrades RNA if the RNA is part of an RNA/DNA duplex. RT uses these two enzymatic activities to convert the single-stranded RNA genome of the virus into a double-stranded DNA that can be integrated into the genome of the host cell. The synthesis of a DNA copy of the viral genome is a crucial step in the life cycle of the virus, and RT has, for that reason, been the target of a number of different anti-HIV drugs (for reviews, e.g., see references 2, 12, 17, 19, 21, 23, 31, and 32). The earliest anti-HIV therapies involved nucleoside RT inhibitors (NRTIs). These analogs enter the cell and are converted to the triphosphate form (nucleoside RT inhibitor triphosphates [NRTI-TPs]) by host cell kinases. Because the NRTI-TPs are analogs of the normal deoxynucleoside triphosphates (dNTPs), NRTI-TPs are incorporated into the primer strand by RT. However, because the NRTI-TPs do not have a 3=-OH group on the sugar or pseudosugar moiety, an NRTI monophosphate (NRTI-MP) that has been incorporated into viral DNA cannot support continued DNA synthesis and the primer chain is terminated. Decreased susceptibility to NRTIs means that the mutant RT has an enhanced ability to select normal dNTPs over the NRTI-TPs. Two primary mechanisms have been identified by which HIV-1 RT becomes less susceptible to the NRTITPs. One mechanism is exclusion, in which the m...

show abstract

“…The cells were transfected with the wild-type pol expression plasmid (lanes 1 to 4 and 14 to 17), pCpol2/M69 (lanes 5, 6, 18, and 19), pCpol2/M73 (lanes 7,8,20, and 21), pCpol2/M83 (lanes 9, 10, 22, and 23), pCpol2/M61 (lanes 11, 12, 24, and 25); lane 13 was derived from cells transfected with pcDNA vector. The pMD9 vector was included in lanes 1,3,4,6,8,10,12,14,16,17,19,21,23, and 25. The transfection cocktail contained the env expression plasmid in all lanes except 1, 13, and 14 and the gag expression plasmid in all lanes except 3, 13, and 16.…”

Section: Protein Requirements For the Encapsidation Of Pol Proteinmentioning

confidence: 99%

“…It has been shown that FV reverse transcriptase (RT) is much more processive than that of orthoretroviruses (31). Furthermore, it has been suggested that a very few molecules of Pol precursor are packaged into the FV capsid (4,31). The FV means of expressing and encapsidating Pol protein has to be viewed in the context of reverse transcription, which appears to occur to a large extent in the virus-producing cell prior to budding (26,32,41).…”

mentioning

confidence: 99%

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Peters

Wiktorowicz

Heinkelein

et al. 2005

J Virol

View full text Add to dashboard Cite

Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76: [10069][10070][10071][10072][10073] 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5 long terminal repeat and one at the 3 end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.Spumaretroviruses, or foamy viruses (FVs), are one of two subfamilies of retroviruses (30). A hallmark of the replication strategy of FVs is the Gag-independent expression of a Pol precursor protein from a spliced RNA (3,6,18,25,40). The consequences of this highly unusual strategy for the generation of a retroviral Pol protein are poorly understood. It has been shown that FV reverse transcriptase (RT) is much more processive than that of orthoretroviruses (31). Furthermore, it has been suggested that a very few molecules of Pol precursor are packaged into the FV capsid (4, 31). The FV means of expressing and encapsidating Pol protein has to be viewed in the context of reverse transcription, which appears to occur to a large extent in the virus-producing cell prior to budding (26,32,41). This leads to linear DNA, which is probably the virion nucleic acid and more relevant for infection than RNA (30).The actual mechanism of FV Pol protein particle incorporation despite lack of a Gag-Pol precursor has remained obscure. It is evident that FVs must have found a means of Pol encapsidation that acknowledges that a Gag-Pol precursor protein, which in orthoretroviruses facilitates Pol incorporation into the viral particle, is not generated (35). We previously showed that the incorporation of (pre)genomic RNA is essential for Pol protein encapsidation (12). Here, we took the experiments a step further and asked whether specific sequences on the RNA which allow Pol protein incorporation can be identified and whether such sequences are different from signals needed to package RNA. Furthermore, we wanted to know what requirements e...

show abstract

Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

Cited by 28 publications

References 40 publications

Foamy Retrovirus Integrase Contains a Pol Dimerization Domain Required for Protease Activation

Foamy Retrovirus Integrase Contains a Pol Dimerization Domain Required for Protease Activation

HIV-1 and HIV-2 Reverse Transcriptases: Different Mechanisms of Resistance to Nucleoside Reverse Transcriptase Inhibitors

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Contact Info

Product

Resources

About