Our results suggest that certain CNS penetrating ARVs used in Neuro-cART regimens may not be effective against HIV-1 infection of astrocytes. 3TC, ZDV and d4T are used in Neuro-cART regimens 
, but here we show that the concentration of these drugs required to achieve virological suppression in astrocytes is 12-, 110, and 187-fold higher than what is achievable in the CSF, respectively. In contrast, 3TC, ZDV and d4T achieved virus inhibition in PBMC and MDM at concentrations that were below or within the respective concentration ranges that are achieved in vivo
. Our results suggest that these NRTIs may not target all the susceptible HIV-1 target cell populations in the CNS, with potential implications for their inclusion in Neuro-cART regimens.
We observed some discordance in EC50 and EC90 values between the astrocyte cell line and primary fetal astrocytes. This highlights some of the limitations of using SVG for astrocyte work and reinforces the need to use the ‘gold standard’ PFA for confirmatory studies. In addition, while the EC50/EC90 values generated here are useful and insightful, they were generated in the context of a single round of infection. Therefore, further studies are required to analyze ARV effectiveness in the context of multiple rounds of infection.
Our recent studies showed that up to approximately 20% of astrocytes can be infected with HIV-1 in vivo
, and therefore astrocytes represent a potentially significant reservoir of HIV-1 in the CNS 
. Astrocytes are critical for maintaining normal brain homeostasis 
,_ENREF_17 and astrocyte dysfunction is known to contribute to HIV-1 neuropathogenesis 
. Continuing to use Neuro-cART regimens containing 3TC, d4T or ZDV could potentially lead to astrocyte infection remaining untargeted, which may contribute to neurocognitive impairment despite virological suppression in plasma. In support of this possibility, the prevalence of HAND is increasing despite suppressive cART 
The current focus on HIV-1 cure and eradication strategies has identified persistently infected viral reservoirs as a major barrier to the successful elimination of the virus 
. These viral reservoirs are located within the brain, gut-associated lymphoid tissue, bone marrow, and genital tract 
. HIV-1 infection of astrocytes is predominantly restricted to the expression of genes encoding the regulatory/accessory HIV-1 proteins 
, some of which are neurotoxic (for example the HIV-1 Tat protein) 
, and contributes to the persistent viral reservoir within the brain that is not cleared by the immune system or ARVs 
. Confirming that the ARVs used in Neuro-cART regimens are active against-, and are present at concentrations sufficient for virological suppression in astrocytes may be important for controlling this potentially significant viral reservoir, and may be necessary to prevent the transcription of HIV-1 genes encoding neurotoxic viral proteins. The optimal targeting of HIV-1 infected astrocytes by ARVs may prevent the expansion of this HIV-1 infected cellular reservoir and potentially aid virus eradication efforts, and may also potentially contribute to better treatment outcomes by reducing neurotoxicity.
While we have shown that both ZDV and d4T have markedly reduced effectiveness in astrocytes compared to macrophages, the underlying mechanism for this remains unknown. All the NRTIs tested here require activation from their inactive native form by three sequential phosphorylation events. In the case of ZDV and d4T, the un-phosphorylated form is converted to the mono-, di- and tri-phosphorylated forms by the cellular thymidine kinases, thymidylate kinase and deoxynucleoside diphosphate kinase, respectively 
. In contrast, 3TC (whose reduced HIV-1 inhibitory activity in astrocytes is relatively modest compared to that of d4T and ZDV) and ABC only have the final phosphorylation event in common, indicating unique cellular kinases are involved in their initial phosphorylation events. The drug is only active against the HIV-1 reverse transcriptase once the drug is tri-phosphorylated, by competing with the natural dNTP substrate for incorporation into DNA and causing chain termination. Three possible explanations could address the reduced effectiveness of d4T and ZDV in astrocytes; differences in cellular uptake of NRTIs, inefficient or incomplete drug activation (due to lower levels of cellular kinases or competition with the natural substrates for the kinases), and inefficient incorporation into DNA (due to higher levels of endogenous nucleotides). To this end, Perno et al.
demonstrated that ZDV was more potent against HIV-1 infection in macrophage lineage cells due to lower levels of competing endogenous thymidine 
, which is a finding that is supported by our experiments when comparing EC90
values of ZDV between PBMC and MDM. Future work will explore this area to better understand the underlying cause of the markedly reduced effectiveness of the d4T and ZDV NRTI thymidine analogues in astrocytes.
In conclusion, the results of our study show that certain NRTIs, in particular the thymidine analogues ZDV and d4T (and to a lesser degree 3TC) have reduced HIV-1 inhibitory astrocytes in vitro compared to macrophage-lineage cells, and furthermore that the inhibitory concentrations of 3TC, ZDV and d4T in astrocytes exceed those achievable in the CSF. Thus, Neuro-cART regimens containing ZDV, d4T and/or 3TC may not effectively target HIV-1 infected astrocytes of the CNS. Furthermore, our results are the first to show differential ARV efficacy in brain cells, thereby introducing an important principle in the future analysis of ARVs.