Deciphering Evolutionary Mechanisms Between Mutualistic and Pathogenic Symbioses.

The continuum between mutualistic and pathogenic symbioses has been an underlying theme for understanding the evolution of infection and disease in a number of eukaryotic-microbe associations. The ability to monitor and then predict the spread of infectious diseases may depend upon our knowledge and capabilities of anticipating the behavior of virulent pathogens by studying related, benign symbioses. For instance, the ability of a symbiotic species to infect, colonize, and proliferate efficiently in a susceptible host will depend on a number of factors that influence both partners during the infection. Levels of virulence are not only affected by the genetic and phenotypic composite of the symbiont, but also the life history, mode(s) of transmission, and environmental factors that influence colonization, such as antibiotic treatment. Population dynamics of both host and symbiont, including densities, migration, as well as competition between symbionts will also affect infection rates of the pathogen as well as change the evolutionary dynamics between host and symbiont. It is therefore important to be able to compare the evolution of virulence between a wide range of mutualistic and pathogenic systems in order to determine when and where new infections might occur, and what conditions will render the pathogen ineffective. This perspective focuses on several symbiotic models that compare mutualistic associations to pathogenic forms and the questions posed regarding their evolution and radiation. A common theme among these systems is the prevailing concept of how heritable mutations can eventually lead to novel phenotypes and eventually new species.

Packaging of heterologous RNAs by a minimal bovine leukemia virus RNA packaging signal into virus particles.

A minimal bovine leukemia virus (BLV) RNA packaging sequence (E) required for heterologous RNAs to be packaged into BLV particles was analyzed. The BLV E was inserted into a non-viral vector, pLacZ, in order to determine if packaging of the non-viral vector RNA would occur. The construct was transfected into cells chronically infected with BLV in order to produce virus particles. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of viral RNA from virus particles revealed that non-viral RNA containing the BLV E was packaged into BLV particles, indicating that the BLV E is necessary and sufficient to allow for packaging of a non-viral vector RNA. We also analyzed the ability of a chimeric murine leukemia virus (MLV) retroviral vector (pLN) containing BLV E to be packaged into BLV particles. Interestingly, it was found that pLNDelta (which does not possess psi+) could be packaged into BLV particles. This indicates that a MLV RNA region outside of psi+ allows for packaging of the MLV RNA into BLV particles.

Interaction of human immunodeficiency virus type 1 Vpr with the HHR23A DNA repair protein does not correlate with multiple biological functions of Vpr.

The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr-HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle.

Virus mutators and antimutators: roles in evolution, pathogenesis and emergence.

Virus mutators (mutant alleles that confer a higher mutant-frequency phenotype than that of the wild type) and antimutators (mutant alleles that confer a lower mutant-frequency phenotype) were discovered in bacteriophage T4 over three decades ago, but there is only limited detailed knowledge about such genetic variants in viruses that infect humans and threaten public health. The creation of mutators and antimutators during the course of viral infection (particularly in the case of RNA viruses) could play a pivotal role in virus evolution, pathogenesis and emergence, and could also frustrate antiviral therapy.

In vivo analysis of human T-cell leukemia virus type 1 reverse transcription accuracy.

Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZalpha peptide gene region (the mutational target). The vector was replicated by trans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 x 10(-6) mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

3'-Azido-3'-deoxythymidine (AZT) and AZT-resistant reverse transcriptase can increase the in vivo mutation rate of human immunodeficiency virus type 1.

How antiretroviral drug resistance influences human immunodeficiency virus type 1 (HIV-1) evolution is not clear. This study tested the hypothesis that antiretroviral drugs such as 3'-azido-3'-deoxythymidine (AZT) can influence the in vivo mutation rate of HIV-1. It was observed that AZT can increase the rate of HIV-1 mutation by a factor of 7 in a single round of replication. In addition, (-)2',3'-dideoxy-3'-thiacytidine (3TC) was also found to increase the mutation rate of HIV-1 by a factor of 3. It was also found that HIV-1 drug-resistant reverse transcriptase (RT) variants can influence the in vivo mutation rate. Replication of HIV-1 with AZT-resistant RTs increased the mutation rate by as much as a factor of 3, while replication of HIV-1 with a 3TC-resistant RT (M184V) had no significant effect on the mutation rate. It was observed that only high-level, AZT-resistant RT variants could influence the in vivo mutation rate (i.e., M41L/T215Y and M41L/D67N/K70R/T215Y). In total, these observations indicate that both antiretroviral drugs and drug resistance mutations can influence the in vivo mutation rate of HIV-1.

The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.

The bovine leukemia virus encapsidation signal is composed of RNA secondary structures.

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5' end of the gag gene (L. Mansky et al., J. Virol. 69:3282-3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the gag start codon) were predicted within the primary signal region, and one stable stem-loop structure (in the gag gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.

The mutation rate of human immunodeficiency virus type 1 is influenced by the vpr gene.

A system has been designed to study the in vivo forward rate of mutation of human immunodeficiency virus type 1 (HIV-1) during one round of replication. A HIV-1 shuttle vector was used that contained the lacZ alpha peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 x 10(-5) mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 reverse transcriptase with sense-strand RNA and DNA templates of the lacZ alpha peptide gene in a cell-free system. To test the hypothesis that the vpr gene product might, at least in part, account for the lower mutation rate observed in vivo, a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-type vpr gene product. A vpr- shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of the vpr- vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence of vpr on the mutation rate. Replication of a vpr- shuttle vector in the presence of a wild-type vpr expression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that the vpr mutation could be complemented in trans. Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence of vpr on the mutation rate.

Forward mutation rate of human immunodeficiency virus type 1 in a T lymphoid cell line.

An in vivo assay was previously developed for detecting forward mutations in human immunodeficiency virus type 1 (HIV-1) in a single cycle of replication. This system uses the lacZalpha peptide gene as a reporter for mutations, and allows for the rates and types of mutations that occur to be determined. The forward mutation rate for HIV-1 in HeLa cells was found to be 3 x 10(-5) mutations per target base pair per cycle. To test whether the mutation rate was influenced by cell type, the mutation rate of HIV-1 in CEM-A cells, a T lymphoid cell line, was determined. The mutation rate of HIV-1 reverse transcription in CEM-A cells was found to be 4 x 10(-5) mutations per target base pair per cycle. The number and types of mutations observed were similar to that in HeLa cells. Specifically, base substitution mutations predominated, and G-to-A transition mutations were the most common base substitution. G-to-A hypermutants were also characterized. The difference in HIV-1 mutation rate between HeLa and CEM-A cells was not significant, indicating that the accuracy of HIV-1 reverse transcription is comparable in both the HeLa and CEM-A cell lines.

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.

Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus.

Genetic variation of the more complex retroviruses in the human T-cell leukemia virus/bovine leukemia virus (HTLV/BLV) group is less than in some other retroviral genera. To test whether reverse transcription of HTLV/BLV group members is less error prone than that of members of other groups, we developed an assay for detecting forward mutations in BLV, similar to that developed for the simpler spleen necrosis virus (SNV). We used this system to study the rates and types of mutations that occur during a single replication cycle. We found that BLV reverse transcription is approximately two and one-half times less error prone than SNV reverse transcription (4.8 x 10(-6) versus 1.2 x 10(-5) mutation per bp per cycle, respectively). The relative numbers of all types of observed mutations (that is, base pair substitutions, frameshifts, deletions, and deletions with insertions) were similar for BLV and SNV.

Molecular basis for virus disease resistance in plants.

Classical studies of virus disease resistance in plants have provided the basis for recent molecular studies of resistance. Three common approaches to the study of resistance have been used. In one approach, nucleotide and/or amino acid sequences of virus strains that overcome disease resistance genes in the host are compared with sequences of strains that do not induce disease in these hosts. In the second approach, resistance/susceptibility of protoplasts is compared with the response of intact plants from which they are derived, to develop hypotheses regarding whether resistance acts at the level of the individual cell or by inhibiting cell-to-cell movement. In the third approach, the mechanism of virus cell-to-cell movement has been studied to clarify one of the basic steps in pathogenesis and to determine the mechanism of disease resistance for certain virus-host interactions.

Genetic engineering of plants for virus resistance.

Historically, control of plant virus disease has involved numerous strategies which have often been combined to provide effective durable resistance in the field. In recent years, the dramatic advances obtained in plant molecular virology have enhanced our understanding of viral genome organizations and gene functions. Moreover, genetic engineering of plants for virus resistance has recently provided promising additional strategies for control of virus disease. At present, the most promising of these has been the expression of coat-protein coding sequences in plants transformed with a coat protein gene. Other potential methods include the expression of anti-sense viral transcripts in transgenic plants, the application of artificial anti-sense mediated gene regulation to viral systems, and the expression of viral satellite RNAs, RNAs with endoribonuclease activity, antiviral antibody genes, or human interferon genes in plants.