Animal models that can be used to study the development of HIV-1 antiviral resistance have the potential to provide important insights relating to the fitness of drug-resistant isolates and the efficacy of inhibitors in different tissues and cell types. To help address this need, we have developed a SIV chimeric virus for infection of pigtail macaques that should be suitable for in vivo analyses of NNRTI therapies. A SIVmne that replicated using HIV-1 RT was as susceptible as HIV-1 to NNRTIs. In contrast, a mutant SIVmne with amino acids 181 and 188 replaced with tyrosine residues displayed partial sensitivity to EFV only.
Both the replacement of specific amino acids at positions 181 and 188 and the replacement of the entire RT coding region of SIVmne
with those of HIV-1 led to approximately a 10-fold reduction in the replication of the viruses. Consistent with these findings, others have reported diminished replication or RT activity in SIVmac239
when amino acids 176 to 190 or 188 alone were changed to the corresponding residues found in the HIV-1 RT (17
). Recent crystal structure analysis of the HIV-2 RT in the presence and absence of NNRTIs suggests that differences in amino acid residues near the active site, including those at positions 181 and 188, could result in significant changes in side chain steric hindrance, electrostatic properties, and positioning of the residues (34
). The substitution of tyrosines at positions 181 and 188 could distort the active site and/or the position of the nucleic acid relative to the active site and affect the activity of the enzyme.
The decrease in replication of RT-SHIVmne
is reminiscent of RT-SHIVmac239
with a compensatory mutation at the viral tRNA-Lys3 PBS (1
). In the absence of this compensatory mutation, the replication of RT-SHIVmac239
appears to be severely impaired. Prior reports indicated that RT-SHIVmac239
was undetectable in CEMx174 cell culture supernatants for up to 65 days in the absence of such a mutation (44
). Because RT-SHIVmne
possesses a canonical tRNA-Lys3 PBS, our results suggest that other adaptive changes may be required for optimal replication of these chimeric viruses.
The reduced replication of RT-SHIVmne
and SIV-RT-YY compared to wild-type SIVmne
is due to diminished RT activity. By quantifying the RT activity in virus from CEMx174 cells, we found that the amount of RT activity per particle of RT-SHIVmne
was approximately 40% less than that of SIVmne
in a quantitative STF-PERT assay. Previous studies have indicated that the polymerase activity of HIV-2 RT, a protein highly homologous to SIV RT, is not significantly different than that of HIV-1 RT in the presence of high levels of dNTPs (14
). This suggests that an RT-SHIVmne
virion, which encodes wild-type HIV-1 RT, contains less mature RT than a wild-type SIVmne
virion. SIV-RT-YY also has a 1.5-fold-lower RT/p27 ratio than SIVmne
, which could reflect either a processing defect or lower enzymatic activity of the mutant RT.
HIV-1 RT cleavage into p66 and p51 subunits in RT-SHIVmne is dependent on SIV protease. The amount of RT subunits in RT-SHIVmac239 had not been measured quantitatively; thus, we examined whether inefficiencies in the folding or processing of RT could account for differences in viral replication efficiency. The reduced replication of RT-SHIVmne did not appear to be due to inefficient cleavage of RT into p66 and p51 subunits by SIV protease, since immunoblot analysis of HIV-1 and RT-SHIVmne particles showed similar ratios of p66 to p51 for the two viruses. However, in the course of this analysis, we noted that the total amount of RT in RT-SHIVmne virions was lower than in HIV-1 virions, which correlated with the lower amount of RT activity of RT-SHIVmne virions compared to wild-type SIVmne virions.
The reduction in the amount of RT in the RT-SHIVmne
particles corresponds to the lower replication of the virus. Previous work in which cells were cotransfected with different ratios of HIV-1 vectors encoding wild-type RT or an RT lacking polymerase activity showed that a decrease in virion particle RT activity caused a significant reduction in replication (19
). When wild-type RT was reduced to 50 or 25% of levels in wild-type particles, the relative infectivity of the virus was 42 and 3%, respectively. RT-SHIVmne
virions appear to incorporate approximately 30% of the RT found in HIV-1 virions, and RT-SHIVmne
replicates to a titer approximately 7% that of wild-type SIVmne
, consistent with the HIV-1 studies.
There are several possible explanations for the reduced amount of RT protein per virion in RT-SHIVmne
. The chimeric p160gag-pol
polyprotein may be expressed inefficiently by reduced frameshifting. The mutant polyprotein could also be folded inefficiently in cells, promoting its degradation. It is also possible that HIV-1 RT could be degraded in RT-SHIVmne
particles by the SIV protease, although RT degradation products were not detected in our immunoblot analyses of RT-SHIVmne
. Alternatively, it is conceivable that the RT-SHIVmne
precursor either does not traffic efficiently to sites of SIV Gag assembly or is less efficiently incorporated into virion particles. Previous studies have reported that there are mutations in HIV-1 RT that interfere with the incorporation and/or processing of p160gag-pol
and that these mutations lead to a reduction in RT activity (16
Normalizing virus in supernatants based on viral RNA content using the same sequence in the pol gene of HIV-1 and RT-SHIVmne has advantages over other approaches for quantifying virions. For instance, ELISA analysis of the CA content of HIV-1 and RT-SHIVmne required the use of different immunological reagents with different sensitivities, and electron microscopy determination of particle counts was difficult because of extensive microvesicle contamination (data not shown). Implicit in the use of real-time RT-PCR detection to quantify the viruses is the assumption that, on average, equal amounts of viral genome RNA copies are present in HIV-1 and RT-SHIVmne particles. Nonetheless, these data clearly show that the ratio of RT to viral genome RNA is different between HIV-1 and RT-SHIVmne.
In spite of their reduced replication, RT-SHIVmne
and SIV-RT-YY showed equivalent sensitivities to HIV-1 and SIVmne
with several NRTIs. As predicted, the presence of HIV-1 RT in SIVmne
susceptible to EFV, NVP, and UC781. RT-SHIVmne
appeared to be slightly more sensitive to EFV, NVP, and UC781 than HIV-1. Hypersensitivity of HIV-1 to NNRTIs has been detected in 17% of patient plasma samples, is more common for EFV than NVP, and is associated with certain NRTI resistance mutations (48
). We did not detect any mutations in RT-SHIVmne
that would explain its increased susceptibility to NNRTIs. NRTIs inhibit reverse transcription by interfering with synthesis of the viral genome, whereas NNRTIs inhibit reverse transcription by binding to the RT enzyme itself. However, the level of RT in a virus particle will affect NNRTI sensitivity. If excess RT is present it can bind the drug, but replication can still occur. In contrast, the target for NRTI inhibition is the viral genome, not RT, and the level of RT will not affect NRTI susceptibility. Thus, the decrease in the amounts of RT in the RT-SHIVmne
virions could make these virions more sensitive to inhibition by NNRTIs but not NRTIs.
Mutation of residues 181 and 188 to tyrosines in SIVmne
increased the sensitivity of the virus to EFV but did not make it as sensitive as HIV-1. This result is in accord with structural data that show that other residues are important in binding these drugs, especially NVP and UC781. Replacement of residues 176 to 190 of RT increased the sensitivity of HIV-2ROD
to NVP to levels three- to fourfold higher than the IC50
value for HIV-1 (18
). Loss of tyrosines at positions 181 and/or 188 of HIV-1 RT reduced the inhibition by first-generation NNRTIs, including NVP (11
). However, the second-generation NNRTI EFV does interact with the tyrosine at 188 in HIV-1 RT, but it has minimal contact with the tyrosine at 181 (15
), which may explain the better efficacy of EFV against SIV-RT-YY. Our data reinforce the notion that the specific tertiary structure of HIV-1 RT targeted by NNRTIs is difficult to recreate even in related molecules.
Selection of NNRTI-resistant RT-SHIVmne
in vitro leads to RT mutations that are seen in patients infected with drug-resistant HIV-1. Mutations commonly associated with NNRTI resistance were seen in RT-SHIVmne
after selection with EFV, NVP, and UC781: K103N, V179D, Y181C, and E194K (2
). It is possible that the unusual P1L mutation that arises in RT-SHIVmne
selected with EFV increases the amount of RT in the virions, by affecting the folding and/or processing of the p160gag-pol
precursor, which would contribute to the resistant phenotype. Using our assay for detection of specific mutations in viral RNA, we can see the proportion of K103N and Y181C resistance mutations increase as RT-SHIVmne
replicates in the presence of escalating doses of EFV and NVP, respectively. These mutations do not appear in virus that is not selected with drugs. This assay will be useful in examining the relative fitness of K103N or Y181C resistance mutations within an animal model in the presence and absence of antiviral selection.
Currently, few studies have been performed in which macaques have been infected with RT-SHIVmac239
and treated with NNRTIs (28
), and only one study evaluated the development of drug resistance mutations in RT after treatment with NVP (49
). More recently, an RT-SHIV based on the SIVmac239
molecular clone was developed that also included the vpr
, and nef
genes from HIV-1NL4-3
). However, replication of this chimeric virus was not detectable in two rhesus macaques 6 and 12 weeks postinoculation, and the susceptibility of this chimera to RT inhibitors was not directly compared to HIV-1 in cell culture. Additional models whose replication and drug-resistant properties have been extensively characterized in vitro may yield more reliable alternatives for in vivo studies. An RT-SHIVmne
/pigtail macaque model that recapitulates the immunopathogenesis, persistence, and systemic distribution of HIV-1 would provide an important tool in the design of clinical treatment regimens to help minimize the development of antiviral drug resistance.