Department of Molecular Virology, Immunology, and Medical Genetics, Center for Retrovirus Research, and Comprehensive Cancer Center, Ohio State University Medical Center, 2078 Graves Hall, 333 West 10th Ave, Columbus, OH 43210, USA1
Ohio State University Biochemistry Graduate Program, Ohio State University, USA2
Molecular, Cellular, and Developmental Biology Graduate Program, Ohio State University, USA3
Author for correspondence: Louis Mansky. Fax +1 614 292 9805. e-mail mansky.3{at}osu.edu
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Abstract |
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Introduction |
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In a single round of HIV-1 replication, a 4-fold increase in the rate of GA transitions in the absence of Vpr was observed, as well as a 4-fold increase in the overall mutation rate when virus was produced from non-dividing cells (Mansky, 1996
). One interpretation of this data was that Vpr directly interacts with RT to influence enzyme fidelity. Another interpretation was that Vpr interacts with other proteins that influence the accuracy of the reverse transcription process. To investigate this mutation phenotype, HIV-1 replication with Vpr mutants containing single amino acid substitutions was analysed in order to map the determinants responsible for the ability of Vpr to influence replication fidelity (Mansky et al., 2000
, 2001
). Vpr mutants were selected based on their ability to interact with two different proteins in the yeast two-hybrid assay, namely UNG and HHR23A, a human homologue of RAD23 derived from yeast (Bouhamdan et al., 1996
, 1998
; Selig et al., 1997
). HHR23A is presumed to function with HHR23B in a nucleotide excision DNA repair pathway as part of a multiprotein complex associated with the xeroderma pigmentosum complementation group C protein (van der Spek et al., 1996
). One Vpr mutant, in which trpyptophan at position 54 was changed to arginine (Vpr*W54R), led to the same mutation phenotype observed during HIV-1 replication in the absence of Vpr (Mansky et al., 2000
).
Vpr*W54R had a phenotype that is comparable to wild-type Vpr in its ability to arrest cells in the G2M phase of the cell cycle, to localize to the nucleus, to be efficiently incorporated into HIV-1 particles and to interact with HHR23A (Selig et al., 1997 ; Mansky et al., 2001
). In contrast, the W54
R substitution prevented Vpr from interacting with UNG and HIV-1 expressing Vpr*W54R led to a 4-fold increase in the rate of G
A mutations. In addition, Vpr*W54R did not allow efficient packaging of UNG into HIV-1 particles. The nuclear form of UNG was preferentially packaged into HIV-1 particles, presumably because both Vpr and UNG are targeted to the nucleus where they could subsequently associate with viral RNA that is destined for packaging into virus particles. This indicates that the interaction and virion-incorporation of Vpr and UNG into HIV-1 particles correlates with the influence of Vpr on the HIV-1 mutation rate. Other studies have also implicated the interaction of UNG with the HIV-1 integrase as an interaction required for UNG incorporation into virus particles (Willetts et al., 1999
). It has not been determined yet if the enzymatic activity of UNG is associated with the mutation rate phenotype. Work with UNG-deficient mice has indicated that the nuclear form of UNG has a specialized role in preventing uracil misincorporation and that another cellular UNG is involved in the removal of uracil in DNA created by cytosine deamination (Nilsen et al., 2000
). Several other Vpr variants, which do not associate with HHR23A showed that the interaction of Vpr with HHR23A is not associated with the ability of Vpr to influence the HIV-1 mutation rate (Mansky et al., 2001
).
A current working hypothesis regarding a possible role for UNG enzymatic activity in HIV-1 replication is that to efficiently remove uracil bases in HIV-1 DNA during synthesis in non-dividing cells, UNG is incorporated into particles via interaction with Vpr (Fig. 2). An intriguing question regarding the incorporation of UNG into HIV-1 particles is whether other repair enzymes are incorporated into virions. The excision of uracil from DNA by UNG will cause abasic (apurinic or apyrimidinic) sites (AP sites). The base excision repair pathway is considered to be the major mechanism to repair AP sites; this pathway is initiated by AP endonuclease (known as HAP, APEX or Ref-1), which catalyses the incision of DNA at AP sites (Friedberg et al., 1995
). Attempts to identify AP endonuclease in HIV-1 particles have failed to date (Mansky et al., 2000
). A DNA ligase activity in HIV-1 particles has yet to be analysed. A lack of enzymes involved in base excision repair (Fig. 1B
) in virus particles may suggest that these enzymes are recruited after virus entry, either in the cytoplasm or, more likely, in the nucleus following integration of viral DNA.
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HIV-1 and related retroviruses (called lentiviruses) can be subdivided into viruses that infect primates (e.g. HIV) and viruses that infect non-primates (e.g. equine infectious anaemia virus, EIAV). Previous work has indicated that EIAV and most non-primate lentiviruses encode and package a dUTPase into virus particles (Table 1) (Elder et al., 1992
). Studies on caprine arthritisencephalitis virus (CAEV) with in-frame nucleotide insertions or deletions in the dUTPase gene have indicated that the replication of dUTPase-minus mutants are severely affected in non-dividing host cells (e.g. primary macrophages) and the virus loads can be decreased 10- to 100-fold in comparison with wild-type virus (Turelli et al., 1997
). The frequency of G
A transition mutations in viral DNA increases during replication of dUTPase-minus CAEV and feline immunodeficiency virus and eventually leads to replication-defective proviruses (Lerner et al., 1995
; Turelli et al., 1997
). Uracil misincorporation into DNA could influence DNA conformation and sequence-specific protein binding and may explain the decrease in virus production and replication. However, replication of dUTPase-minus CAEV mutants in dividing cells (e.g. mitogen-stimulated T-cells and continuous T-cell lines) is only minimally decreased, suggesting that actively dividing cells could have sufficiently high endogenous dUTPase activity, which compensates for the lack of virion-associated dUTPase activity (Turelli et al., 1997
).
Herpesviruses, poxviruses and UNG
UNG is encoded and expressed by DNA viruses of two main families, the Herpesviridae and the Poxviridae (Table 1). Herpesviruses replicate their viral DNA and assemble virus capsids in the nucleus of infected cells. The ability of UNG to influence virus replication of different herpesviruses has implied a role for viral UNG in the replication of virus in the host, particularly in non-dividing cells (e.g. terminally differentiated cells), where levels of cellular UNG are believed to be low. Viral UNG has been shown to be dispensable for replication in cell culture (Mullaney et al., 1989
) but herpes simplex virus type 1 (HSV-1) UNG-minus mutants replicated and spread poorly in mice (Pyles & Thompson, 1994b
).
A more recent study suggests that the elimination of both viral and cellular UNG activity does not affect the efficiency of replication for another herpesvirus, varicella-zoster virus (VZV) (Reddy et al., 1998 ). A VZV mutant with a deletion of the gene encoding UNG was shown to replicate as efficiently as the parental virus in cell culture. A natural inhibitor of UNG from the Bacillus subtilis bacteriophages PBS1 and PBS2 (uracil-DNA glycosylase inhibitor, UGI) inactivates UNG activity from a variety of organisms, including herpesviruses. The replication of UGI-expressing VZV, in either the presence or the absence of viral UNG, was as efficient as that in the parental virus. This implies that cellular UNG cannot functionally replace viral UNG when it is not expressed. This provides an indication that UNG may be dispensable for replication in actively dividing cells in culture.
Human cytomegalovirus (CMV) UNG was first reported to delay viral DNA synthesis and replication (Prichard et al., 1996 ). More recent studies have suggested that UNG excises uracil residues from replicating CMV DNA to create sites that can serve as substrates for initiation of recombination-dependent replication late in infection (Courcelle et al., 2001
). CMV DNA replication is thought to switch from a bidirectional (theta structure) mode early in infection to a rolling-circle mode of replication late in the infection process. The nicks in the DNA generated by the removal of uracil residues by UNG and cleavage of AP sites by AP endonuclease could serve a functional role in the switch from bidirectional to rolling-circle replication (Fig. 3
). In quiescent, non-dividing cells, the lack of CMV UNG expression leads to a delay in replication for 48 h (Courcelle et al., 2001
). In actively dividing cells, virus replication of the UNG mutant occurs without delay (Courcelle et al., 2001
). This indicates that the role of virus-encoded UNG is particularly important in non-dividing cells. In summary, studies with herpesvirus UNG indicate that there is some debate as to whether viral UNG is important for virus replication in dividing cells; viral UNG appears to play an important role for replication in non-dividing cells.
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Herpesviruses and poxviruses encode dUTPase (Table 1), as do some other DNA viruses (Baldo & McClure, 1999
) such as African swine fever virus (ASFV) (Dixon et al., 1994
; Yanez et al., 1995
). ASFV is a member of the Asfivirus genus of the family Asfarviridae and has some similarities to poxviruses. ASFV mutants that do not express dUTPase have been associated with inefficient replication in non-dividing cells (Oliveros et al., 1999
). Herpesvirus dUTPase-minus mutants have been reported to be attenuated for neurovirulence, lack the ability to reactivate virus replication from latency (Pyles et al., 1992
) and possess an increased frequency of mutant formation (Pyles & Thompson, 1994a
).
Orf virus is a member of the Parapoxvirus genus of the family Poxviridae. The orf virus NZ2 strain deletion variant, isolated after serial passage in primary bovine testis cells, had a deletion of the E3L gene, which encodes a protein related to the HSV dUTPase, indicating that this gene is dispensable for replication in dividing cells (Fleming et al., 1995 ). However, an attenuated variant of the orf virus D1701 strain, which is used as a live vaccine against contagious ecthyma in sheep, contains an intact E3L gene (Cottone et al., 1998
). This gene has been shown recently to encode a functional dUTPase (Cottone et al., 2002
).
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Concluding remarks |
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Acknowledgments |
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References |
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