Studies of FIV, SIV, and HIV have demonstrated that the pathogenicity of the infecting lentiviral strain plays an important role in determining the viral load and the severity of host immunodeficiency (22
). However, the specific viral characteristics associated with pathogenicity remain relatively poorly understood. FIV infection of the domestic cat offers a model system for research on lentiviral pathogenesis in an authentic host species with a disease pathology highly similar to that of HIV/AIDS, since one of the best-characterized features of the FIV system is the exhaustive documentation of strain-specific pathologies associated with genetically divergent FIV strains (5
). Therefore, studies on the viral genetic basis for FIV pathogenicity can inform our understanding of other lentiviruses, such as HIV, by examining important genetic elements which lentiviruses share, such as Vif. Likewise, elements that are unique to FIV, such as OrfA, can be informative at a comparative level in understanding lentiviral strategies for replication and evasion of host defenses.
In this study, we examined the basis for FIV pathogenicity using vif
accessory gene chimeras between two FIV molecular clones with reproducibly and strikingly contrasting disease potentials: HV-FIV (FIV-C36), which is highly virulent in vivo
and replicates rapidly in vitro
; and LV-FIV (FIV-PPR), which is less virulent in vivo
and replicates more slowly and/or to lower titers in vitro
. We used overlapping PCR (82
) to clone vif
accessory gene chimeras. The generation of such chimeras is feasible in FIV but not in human or primate lentiviruses, which contain overlapping transcription reading frames in accessory and structural genes () (94
We generated 3 chimeric FIVs in which vif, orfA, or vif/orfA from HV-FIV was substituted for these genes on the LV-FIV backbone, producing FIV-HVvif, FIV-HVorfA, and FIV-HVvif/orfA, respectively. In all cell systems evaluated and by all measures of viral replication, FIV-HVorfA displayed moderately increased replication kinetics compared to the replication of the isogenic parental LV-FIV, and chimeric viruses expressing HV-FIV vif or vif/orfA had strongly increased replication kinetics compared to the replication of LV-FIV, nearly equaling that of the virulent parental FIV. This finding suggests that these nonstructural elements may play a significant role in the capacity for replication and, thus, may be a mechanism for enhancing virulence.
In Mya-1 cells, the degree of viral replication for chimeric viruses (versus the replication of the parental LV-FIV, as measured by p26 ELISA  or RT activity ) appeared to be relatively more enhanced than the viral DNA load (as measured by qPCR ). While the assays used cannot be directly compared and the scales of comparison are different (linear for viral production and log for proviral integration), this observation suggests that high-virulence Vif and OrfA may have a greater enhancement of postintegration events (viral transcription, translation, assembly, and release) than of preintegration events. This observation is consistent with a number of possible mechanisms, including an enhanced ability to counteract deamination-dependent inhibition by feline A3.
We further demonstrated that the enhancement of FIV-HVvif replication is apparent only in the presence of feline A3 proteins, suggesting that naturally occurring differences in lentiviral Vif contribute to viral replication capacity and virulence via interaction with host A3. The interaction between lentiviral Vif and host A3 is a critical factor determining the species specificity of lentiviruses. For instance, the inability of HIV-1 to infect species other than humans and chimpanzees is based largely on the species-specific adaptation of Vif to human A3 proteins (72
). Likewise, we have found that challenge of domestic cats with the puma strain of FIV (FIV-Pco) results in an initial productive infection that diminishes to undetectable levels over time (98
). The reductions in virus expression coincide with G-to-A hypermutation in the provirus, consistent with the interpretation that FIV-Pco Vif is not well adapted to target domestic cat A3 (99
While pathogenicity is likely related to numerous complex viral traits, the ability to counteract A3 intracellular restriction via a more “robust” Vif may be an important virulence determinant. Vif from one particular lentiviral species typically protects against A3 proteins from its native host; i.e., FIV Vif mitigates feline A3 activity, HIV Vif mitigates human A3 activity, etc. Vif antagonism of a matched-host A3 protein is not necessarily complete, however. A3 proteins can cause sublethal levels of G-to-A mutation even in the presence of a functional Vif (100
), suggesting that sufficient A3 evades Vif-mediated degradation to directly affect virus evolution/divergence. Additionally, when transfected at high levels, A3 proteins are able to overwhelm matched-host Vif, resulting in a virus restriction phenotype compared to the phenotype seen with normal A3 expression (55
). In a clinical setting, polymorphisms in human A3 genes have been associated with the risk of HIV acquisition (102
) and with HIV disease progression (102
). Likewise, several reports have found that higher levels of A3 expression are associated with lower HIV loads (106
) and resistance to infection (108
). These studies suggest that there is a dynamic balance between A3-mediated restriction and Vif-mediated protection that logically extends to a role for lentiviral Vif with significant impact on viral replication and pathogenicity. Indeed, it has been shown that the ability of HIV Vif to neutralize A3 proteins in vitro
can differ between HIV subtypes (110
) or between strains of the same subtype (112
While the studies cited above utilized in vitro
single-replication-cycle assays to support these observations, differences in Vif activity between naturally occurring lentiviral strains have not been explored in depth, especially in the context of multicycle whole-virus replication. Here, we examined fully replication-competent vif
gene chimeras constructed between two FIV strains with well-characterized and divergent pathological consequences that directly correlate with in vitro
growth characteristics (47
). These results suggest a model in which lentivirus-host adaptation is a continuum that is highly dependent upon Vif and that naturally occurring differences in lentiviral Vif can control the virus replication that is associated with pathogenic potential. Explanations for these findings might include strain-specific differences in (i) vif
transcription levels, (ii) Vif-induced degradation of A3, (iii) Vif protein stability, or (iv) sequestration of A3 proteins which does not result in degradation.
The finding that HV-FIV Vif confers greater replication capacity than LV-FIV Vif in a common viral backbone additionally raises the question of what regions of Vif are essential for conferring this phenotype. Comparison of the Vif amino acid sequences of these two viruses shows 84% identity, with 40 amino acid differences, 10 of which are considered amino acids with highly dissimilar properties (see Fig. S2 in the supplemental material). Amino acid changes occur in a putative Cullin5 zinc-coordinating motif and immediately adjacent to a putative BC box (proposed by Stern et al. [72
]), both considered highly relevant functional domains. Alternatively, sequence differences which affect the Vif expression level or stability might be relevant.
Multiple potential functions have been ascribed to the small, 77-amino-acid FIV OrfA protein. Originally characterized as a transactivator of viral protein expression (77
), similar to HIV Tat, the mechanism of action is distinct from those of other lentiviral transactivators and the net upregulation of transcription is relatively weak (73
). OrfA has also been shown to affect virion formation and infectivity (73
), as well as to localize to the nucleus and induce G2
cell cycle arrest (78
), similar to HIV Vpr. Most recently, it has been demonstrated that OrfA downregulates the surface expression but not the transcription or translation of the primary FIV receptor CD134 and that OrfA-negative FIVs are unable to productively infect CD134-expressing cells (80
). OrfA inhibition of CD134 may facilitate more-efficient virus release due to decreased receptor interactions with progeny virus, similar to the effects attributed to HIV Vpu and Nef on CD4 (90
). However, another recent study suggests that this effect may be related to cell type and, potentially, other virus-specific factors (89
). Interestingly, the orfA
gene is the most genetically heterogeneous portion of the FIV genome when compared across all known FIV isolates. HV-FIV and LV-FIV OrfA proteins share only 65% amino acid identity, with 27 total differences, of which 8 are highly dissimilar amino acids (see Fig. S2 in the supplemental material). This high level of diversity provides a potential basis for functional differences between strains.
We found that replacing the LV-FIV orfA
with the HV-FIV orfA
resulted in a moderate increase in viral replication capacity. The double accessory gene chimera FIV-HVvif/orfA displayed replication kinetics similar to those of FIV-HVvif (, , and ), suggesting that the gain of function resulting from the high-virulence orfA
is weaker than that conferred by the high-virulence vif
. FIV OrfA-mediated downregulation of the FIV primary binding receptor CD134 is associated with the ability of the virus to replicate in CD134-expressing cells (80
). Thus, a possible mechanism to explain the replication differences between strains could be that increased CD134 downregulation might facilitate efficient virus release and promote replication. We observed that CD134 surface expression appeared to correlate inversely with the rate of virus replication; i.e., lower expression of CD134 was associated with more-rapid viral replication (). However, ascribing effects on cellular protein expression to different viruses in a low-MOI, multiple-replication-cycle experiment is likely to be confounded by viruses replicating and spreading to new cells at different rates due to potentially unrelated elements (such as Vif).
In this study, we chose to focus on a comparison of vif
chimeric and parental strain replication in T cells and PBMC, since these are major targets of FIV infection and replication in these cell types has been shown to correlate with replication in vivo
). However, macrophages also play an important role in establishing productive lentiviral infection, and both vif
are required for productive FIV infection of macrophages (49
). Therefore, future work aimed at understanding the mechanism of the observed differences in vif
chimeric virus replication may benefit from comparison of virus replication in feline macrophages.
The results of this study identify an important mechanism for lentiviral virulence in a model relevant for HIV/AIDS. While it is well established that A3 plays a key role in restricting lentiviral cross-species infection, these findings support the concept that host cellular A3 can also affect the replication of host-adapted lentiviral strains, resulting in a continuum of consequences from aborted infection to significant virulence. The dynamic balance between A3-driven host restriction and Vif-driven viral escape from restriction is ongoing and may influence the ability of lentiviruses to cause progressive disease. This system will provide an opportunity to study specific Vif-A3 interactions that are relevant in vivo and determine the mechanism of increased Vif-dependent lentiviral replication capacity, suggesting rational and novel modalities for additional lentiviral therapeutics. The chimeric system outlined here will also provide a useful basis for more clearly delineating the function of OrfA with respect to FIV replication and virulence and for potentially identifying additional host restriction factors relevant to limiting lentiviral pathogenicity.